bsa  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Name:
    BSA Molecular Biology Grade
    Description:
    BSA Molecular Biology Grade 12 mg
    Catalog Number:
    b9000s
    Price:
    30
    Size:
    12 mg
    Category:
    Buffers
    Buy from Supplier


    Structured Review

    New England Biolabs bsa
    BSA Molecular Biology Grade
    BSA Molecular Biology Grade 12 mg
    https://www.bioz.com/result/bsa/product/New England Biolabs
    Average 90 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    bsa - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) Product Images from "Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination"

    Article Title: Integrin-linked kinase is required for laminin-2-induced oligodendrocyte cell spreading and CNS myelination

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200304154

    LN-2 activates ILK activity and DN-ILK inhibits oligodendrocyte cell spreading. (A) OPCs were transfected with 10 MOI vector alone as a control or with DN-ILK (kinase-inactive form of ILK) by adenovirus gene transfer. After 24 h, transfectants were plated on LN-2 in serum-free media with N 2 and cultured for the indicated time periods. ILK was immunoprecipitated from cell extracts. ILK activity was determined with MBP as an exogenous substrate. Total amounts of ILK proteins in the immunoprecipitates were detected by immunoblot with an anti-ILK antibody. Results indicate that LN-2 stimulates ILK activity. (B) 1 h after cells were plated on BSA, LN-2, TSP-1, and TN-C. ILK activity was detected by phosphorylation of MBP. ILK levels were detected as controls. LN-2 stimulates ILK activity more effectively than any of the other ECMs at 1h (P
    Figure Legend Snippet: LN-2 activates ILK activity and DN-ILK inhibits oligodendrocyte cell spreading. (A) OPCs were transfected with 10 MOI vector alone as a control or with DN-ILK (kinase-inactive form of ILK) by adenovirus gene transfer. After 24 h, transfectants were plated on LN-2 in serum-free media with N 2 and cultured for the indicated time periods. ILK was immunoprecipitated from cell extracts. ILK activity was determined with MBP as an exogenous substrate. Total amounts of ILK proteins in the immunoprecipitates were detected by immunoblot with an anti-ILK antibody. Results indicate that LN-2 stimulates ILK activity. (B) 1 h after cells were plated on BSA, LN-2, TSP-1, and TN-C. ILK activity was detected by phosphorylation of MBP. ILK levels were detected as controls. LN-2 stimulates ILK activity more effectively than any of the other ECMs at 1h (P

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Cell Culture, Immunoprecipitation

    LN-2 promotes oligodendrocyte cell spreading in vitro. (A) Purified OPCs isolated from the rat forebrain were cultured in the presence of PDGF and basic FGF for 3 d. Cells were fixed after three further days of growth on BSA, LN-2, TSP-1, and TN-C in serum-free media with N 2 supplement (N 2 ). Cells were plated and stained for O1 (left, green) and DAPI (blue). Bar, 100 μm. (B) The relative percentages of O1 + cells with broad myelin membrane sheets on different ECM substrates were scored. LN-2 increases the ratio of O1 + cells with myelin membrane sheets to the total number of O1 + cells. Results are presented as ±SEM; and comparisons by ANOVA are significant at P
    Figure Legend Snippet: LN-2 promotes oligodendrocyte cell spreading in vitro. (A) Purified OPCs isolated from the rat forebrain were cultured in the presence of PDGF and basic FGF for 3 d. Cells were fixed after three further days of growth on BSA, LN-2, TSP-1, and TN-C in serum-free media with N 2 supplement (N 2 ). Cells were plated and stained for O1 (left, green) and DAPI (blue). Bar, 100 μm. (B) The relative percentages of O1 + cells with broad myelin membrane sheets on different ECM substrates were scored. LN-2 increases the ratio of O1 + cells with myelin membrane sheets to the total number of O1 + cells. Results are presented as ±SEM; and comparisons by ANOVA are significant at P

    Techniques Used: In Vitro, Purification, Isolation, Cell Culture, Staining

    Oligodendrocyte cell spreading promoted by LN-2 requires PI3K, not MAPK. (A) 45 min and 6 h after cells were plated on BSA, LN-2, TSP-1 and TN-C, phosphorylation of AKT was detected by immunoblot with AKT phospho-ser473 (pAKT) antibody. AKT levels were detected as controls. (B–D) The blocking effects of both 0.01 μM wortmannin (wort) and 0.5 μM LY294002 (PI3K inhibitors) on pAKT or of 4 μM U0126 (MAPK/ERK inhibitor) on pMAPK at 15 and 45 min were shown by immunoblot. AKT or MAPK levels were detected as controls. (E) Cells were fixed after 3 d growth on LN-2 with various inhibitors at the same concentrations used in B–D, after pretreatment for 5 min before plating and staining by O1 (green) and DAPI (blue). Bar, 100 μm. (F) OPCs were plated on LN-2 and infected with GFP-vector (20 MOI) alone or a DN-AKT (10 MOI) in serum-free media with N 2 for 3 d by an adenovirus-mediated tetracycline (Tet)–off inducible system. Double staining with O1 (left, red) and GFP (left, green) or with O1 (middle and right, green) and HA-epitope–tagged DN-AKT (middle and right, red) delineates cells infected by vector or DN-AKT. Incubation of tetracycline responsive promoter (TRE)-DN-AKT (10 MOI) with tetracycline-controlled transactivator (tTA, 10 MOI) blocks LN-2–induced cell spreading by expression of DN-AKT. Incubation with 10 μg/ml Tet turned off the expression of DN-AKT, showing cell spreading as much as GFP-vector alone. Bar, 100 μm. (G) The relative percentage of O1 + cells with broad myelin membrane sheets on LN-2 with various inhibitors is shown. Treatment with PI3K inhibitors reduces myelin membrane formation up to 50% (P
    Figure Legend Snippet: Oligodendrocyte cell spreading promoted by LN-2 requires PI3K, not MAPK. (A) 45 min and 6 h after cells were plated on BSA, LN-2, TSP-1 and TN-C, phosphorylation of AKT was detected by immunoblot with AKT phospho-ser473 (pAKT) antibody. AKT levels were detected as controls. (B–D) The blocking effects of both 0.01 μM wortmannin (wort) and 0.5 μM LY294002 (PI3K inhibitors) on pAKT or of 4 μM U0126 (MAPK/ERK inhibitor) on pMAPK at 15 and 45 min were shown by immunoblot. AKT or MAPK levels were detected as controls. (E) Cells were fixed after 3 d growth on LN-2 with various inhibitors at the same concentrations used in B–D, after pretreatment for 5 min before plating and staining by O1 (green) and DAPI (blue). Bar, 100 μm. (F) OPCs were plated on LN-2 and infected with GFP-vector (20 MOI) alone or a DN-AKT (10 MOI) in serum-free media with N 2 for 3 d by an adenovirus-mediated tetracycline (Tet)–off inducible system. Double staining with O1 (left, red) and GFP (left, green) or with O1 (middle and right, green) and HA-epitope–tagged DN-AKT (middle and right, red) delineates cells infected by vector or DN-AKT. Incubation of tetracycline responsive promoter (TRE)-DN-AKT (10 MOI) with tetracycline-controlled transactivator (tTA, 10 MOI) blocks LN-2–induced cell spreading by expression of DN-AKT. Incubation with 10 μg/ml Tet turned off the expression of DN-AKT, showing cell spreading as much as GFP-vector alone. Bar, 100 μm. (G) The relative percentage of O1 + cells with broad myelin membrane sheets on LN-2 with various inhibitors is shown. Treatment with PI3K inhibitors reduces myelin membrane formation up to 50% (P

    Techniques Used: Blocking Assay, Staining, Infection, Plasmid Preparation, Double Staining, Incubation, Expressing

    2) Product Images from "Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch"

    Article Title: Enhanced activity of adenine-DNA glycosylase (Myh) by apurinic/apyrimidinic endonuclease (Ape1) in mammalian base excision repair of an A/GO mismatch

    Journal: Nucleic Acids Research

    doi:

    Stimulation of Myh binding to non-cleavable T/GO substrate by Ape1 and H309N. The binding reactions were carried out at 37°C for 10 min with 50 pM T/GO DNA in the presence of Myh, Myh/Ape1, Myh/H309N or Myh/BSA at the concentrations indicated. The binding products were analyzed by EMSA on an 8% native polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left.
    Figure Legend Snippet: Stimulation of Myh binding to non-cleavable T/GO substrate by Ape1 and H309N. The binding reactions were carried out at 37°C for 10 min with 50 pM T/GO DNA in the presence of Myh, Myh/Ape1, Myh/H309N or Myh/BSA at the concentrations indicated. The binding products were analyzed by EMSA on an 8% native polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left.

    Techniques Used: Binding Assay, Labeling

    Stimulation of Myh–DNA complex formation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 10 min with 0.2 nM A/GO substrate and 0.5 nM Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. One aliquot of each reaction was directly analyzed by EMSA on an 8% native polyacrylamide gel. The other aliquot, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. ( A ) Phosphoimage of Myh–DNA complexes. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left. ( B ) Phosphoimage of accumulation of glycosylase product. The intact 96mer DNA (Intact) and cleaved 60mer product (Cleaved product) are indicated on the left.
    Figure Legend Snippet: Stimulation of Myh–DNA complex formation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 10 min with 0.2 nM A/GO substrate and 0.5 nM Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. One aliquot of each reaction was directly analyzed by EMSA on an 8% native polyacrylamide gel. The other aliquot, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. An asterisk indicates the 5′-end-labeled strand. ( A ) Phosphoimage of Myh–DNA complexes. The observed bands a and b are marked on the right. The bound and free DNA forms are indicated on the left. ( B ) Phosphoimage of accumulation of glycosylase product. The intact 96mer DNA (Intact) and cleaved 60mer product (Cleaved product) are indicated on the left.

    Techniques Used: Mutagenesis, Labeling

    Glycosylase stimulation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO substrate and increasing concentrations of Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. Then each reaction mixture, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. A plot of the fraction of glycosylase product versus [Myh] is shown.
    Figure Legend Snippet: Glycosylase stimulation by Ape1 and H309N. The glycosylase reactions were carried out at 37°C for 30 min with 0.2 nM A/GO substrate and increasing concentrations of Myh in the presence of 50 nM wild-type Ape1 (wt Ape1), 50 nM catalytic mutant H309N (H309N mutant), 50 nM BSA or buffer. Then each reaction mixture, with alkaline treatment, was analyzed on a 15% denaturing polyacrylamide gel. A plot of the fraction of glycosylase product versus [Myh] is shown.

    Techniques Used: Mutagenesis

    3) Product Images from "Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies"

    Article Title: Applying Unconventional Secretion in Ustilago maydis for the Export of Functional Nanobodies

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18050937

    Chitin-binding activity of recombinant Cts1. ( A ) SDS-PAGE analysis of fractions obtained from IMAC purification of His-tagged Cts1 (Cts1 H ) produced in Escherichia coli . CE, cell extract; FT, flow through; W, wash step; E, elution fractions using different concentrations of imidazole (subscripts). Cts1 H is depicted with a black arrowhead; ( B ) Chitin activity assay with distinct amounts of IMAC purified Cts1 H . Activity was determined by monitoring the change in fluorescence using the fluorogenic chitinase substrate 4-methylumbelliferyl β- d - N , N ′, N ′′-triacetylchitotrioside (4-MUC). RFU, relative fluorescence units; ( C ) Chitin-binding activity of distinct amounts (5 or 10 µg) of purified Cts1 H (indicated by black arrowhead). Equal amounts of purified BSA (NEB, Ipswich, MA, USA) were used as a negative control (depicted by open arrowhead). Chitin Magnetic Beads were mixed with respective proteins (IN), washed rigorously and bound protein was eluted using Laemmli buffer (OUT).
    Figure Legend Snippet: Chitin-binding activity of recombinant Cts1. ( A ) SDS-PAGE analysis of fractions obtained from IMAC purification of His-tagged Cts1 (Cts1 H ) produced in Escherichia coli . CE, cell extract; FT, flow through; W, wash step; E, elution fractions using different concentrations of imidazole (subscripts). Cts1 H is depicted with a black arrowhead; ( B ) Chitin activity assay with distinct amounts of IMAC purified Cts1 H . Activity was determined by monitoring the change in fluorescence using the fluorogenic chitinase substrate 4-methylumbelliferyl β- d - N , N ′, N ′′-triacetylchitotrioside (4-MUC). RFU, relative fluorescence units; ( C ) Chitin-binding activity of distinct amounts (5 or 10 µg) of purified Cts1 H (indicated by black arrowhead). Equal amounts of purified BSA (NEB, Ipswich, MA, USA) were used as a negative control (depicted by open arrowhead). Chitin Magnetic Beads were mixed with respective proteins (IN), washed rigorously and bound protein was eluted using Laemmli buffer (OUT).

    Techniques Used: Binding Assay, Activity Assay, Recombinant, SDS Page, Purification, Produced, Flow Cytometry, Fluorescence, Negative Control, Magnetic Beads

    4) Product Images from "The fragile X mental retardation protein inhibits translation via interacting with mRNA"

    Article Title: The fragile X mental retardation protein inhibits translation via interacting with mRNA

    Journal: Nucleic Acids Research

    doi:

    Dose-dependent reduction of translation of brain poly(A) RNA but not rabbit reticulocyte poly(A) RNA by FMRP. ( A ) Dose-dependent effect of FMRP. An aliquot of 1.5 µg poly(A) RNA was used in each reaction. Translation yield for each reaction is represented by the percentage of TCA precipitable counts (c.p.m.) obtained from mock-treated reactions. At 10 min after initiation of translation yields for reactions exposed to various amounts of FMRP or BSA as depicted at the bottom were subjected to one way ANOVA analysis ( P
    Figure Legend Snippet: Dose-dependent reduction of translation of brain poly(A) RNA but not rabbit reticulocyte poly(A) RNA by FMRP. ( A ) Dose-dependent effect of FMRP. An aliquot of 1.5 µg poly(A) RNA was used in each reaction. Translation yield for each reaction is represented by the percentage of TCA precipitable counts (c.p.m.) obtained from mock-treated reactions. At 10 min after initiation of translation yields for reactions exposed to various amounts of FMRP or BSA as depicted at the bottom were subjected to one way ANOVA analysis ( P

    Techniques Used:

    5) Product Images from "Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks"

    Article Title: Damage-induced lncRNAs control the DNA damage response through interaction with DDRNAs at individual double-strand breaks

    Journal: Nature cell biology

    doi: 10.1038/ncb3643

    Sequence-specific localization of DDRNAs at DNA damage sites is transcription-dependent. ( A ) Images of NIH2/4 cells expressing GFP-LacR, microinjected with double-stranded DDRNA-Cy5, artificial CXCR4-Cy5 miRNA (Ctrl RNA 1) or let-7a-Cy5 miRNA (Ctrl RNA 2), together with BSA (-) or I-SceI restriction enzyme (+) and imaged 4 h post injection. Scale bar 5 µm. Inset is a magnified view of the boxed region. Images from one out of 3 experiments with similar results. ( B ) Quantification of (A) showing the number of fluorophore-labeled RNA molecules at the locus as measured by single-molecule analysis based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( C ) DDRNAs localize at the damage site to restore DDR focus formation. NIH2/4 cells knocked-down for Dicer and Drosha were mildly permeabilized and incubated with DDRNA-Cy5 or CXCR4-Cy5 (Ctrl RNA 1). The bar plot shows the percentage of cells positive for co-localization of 53BP1 with TetR, of RNA-Cy5 with TetR and the triple co-localization of 53BP1, RNA-Cy5 and TetR. Error bars indicate SEM (for siLuc and siDic n=4, for siDro n=3 independent experiments, ≥70 cells analysed in total per condition). ( D ) NIH2/4 cells expressing YFP-TetR and inducible I-SceI were treated with AM, DRB or ACTD at low and high doses or vehicle alone for 2 h before cut induction, then mildly permeabilized and incubated with DDRNA-Cy5. The bar plots show the percentage of cells in which DDRNA signal co-localizes with the TetR spot. Error bars indicate SEM (n=3 independent experiments, ≥80 cells analysed in total per condition). ( E ) NIH2/4 cells expressing GFP-LacR were microinjected with double-stranded DDRNA-Cy5, together with I-SceI protein and AM and imaged 4 h post injection. The plot shows the number of DDRNA molecules at the locus as measured by single-molecule counting based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( B,E ) P values were calculated using two-tailed t-test. ( C,D ) P values were calculated using chi-squared test. *** P
    Figure Legend Snippet: Sequence-specific localization of DDRNAs at DNA damage sites is transcription-dependent. ( A ) Images of NIH2/4 cells expressing GFP-LacR, microinjected with double-stranded DDRNA-Cy5, artificial CXCR4-Cy5 miRNA (Ctrl RNA 1) or let-7a-Cy5 miRNA (Ctrl RNA 2), together with BSA (-) or I-SceI restriction enzyme (+) and imaged 4 h post injection. Scale bar 5 µm. Inset is a magnified view of the boxed region. Images from one out of 3 experiments with similar results. ( B ) Quantification of (A) showing the number of fluorophore-labeled RNA molecules at the locus as measured by single-molecule analysis based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( C ) DDRNAs localize at the damage site to restore DDR focus formation. NIH2/4 cells knocked-down for Dicer and Drosha were mildly permeabilized and incubated with DDRNA-Cy5 or CXCR4-Cy5 (Ctrl RNA 1). The bar plot shows the percentage of cells positive for co-localization of 53BP1 with TetR, of RNA-Cy5 with TetR and the triple co-localization of 53BP1, RNA-Cy5 and TetR. Error bars indicate SEM (for siLuc and siDic n=4, for siDro n=3 independent experiments, ≥70 cells analysed in total per condition). ( D ) NIH2/4 cells expressing YFP-TetR and inducible I-SceI were treated with AM, DRB or ACTD at low and high doses or vehicle alone for 2 h before cut induction, then mildly permeabilized and incubated with DDRNA-Cy5. The bar plots show the percentage of cells in which DDRNA signal co-localizes with the TetR spot. Error bars indicate SEM (n=3 independent experiments, ≥80 cells analysed in total per condition). ( E ) NIH2/4 cells expressing GFP-LacR were microinjected with double-stranded DDRNA-Cy5, together with I-SceI protein and AM and imaged 4 h post injection. The plot shows the number of DDRNA molecules at the locus as measured by single-molecule counting based on stepwise photobleaching. Dots represent individual cells. The black line represents the mean ± SEM (data are shown as pool of n=3 independent experiments). ( B,E ) P values were calculated using two-tailed t-test. ( C,D ) P values were calculated using chi-squared test. *** P

    Techniques Used: Sequencing, Expressing, Injection, Labeling, Incubation, Single Molecule Counting, Two Tailed Test

    6) Product Images from "PIE-1 Translation in the Germline Lineage Contributes to PIE-1 Asymmetry in the Early Caenorhabditis elegans Embryo"

    Article Title: PIE-1 Translation in the Germline Lineage Contributes to PIE-1 Asymmetry in the Early Caenorhabditis elegans Embryo

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.118.200744

    Quantification of the increase in PIE-1::GFP concentration in the P lineage. A. Schematic of PIE-1 (gray) localization from the 1-cell to the 4-cell stage. Maternally deposited PIE-1 segregates asymmetrically to the germline blastomeres P1 and P2 during the first two rounds of cell division. PIE-1 is also degraded in somatic cells. Sister cells are connected by a line. B. Top panel: Coomassie stained SDS-PAGE gel of recombinant GFP and BSA, which was used as a loading standard. Bottom panels: Images of N2 and PIE-1::GFP embryos bathed in 300 nM GFP. Images were pseudocolored using the CyanHot lookup table in ImageJ (scale at the bottom). In order to highlight the dimmer PIE-1::GFP signals, the nuclear signal in the main image of the 4-cell embryo is saturated. The image normalization was adjusted equivalently in the 2 and 4-cell embryo insets such that the nuclear signal is not saturated. PIE-1::GFP concentration in the 1-cell embryo was determined using a 150 nM GFP bath, but is shown in a bath of 300 nM GFP to allow comparison with the later stage embryos. C. Estimates of PIE-1::GFP concentration in P0, P1 and P2. For P1 and P2, concentration estimates are shown for the entire cell (Tot), the cytoplasm (Cyt) and for the nucleus (Nuc). Mean concentrations and the number of embryos analyzed are indicated below the graph. Error bars represent 95% confidence intervals. Statistical significance was determined using unpaired t -tests with Welch’s correction for comparisons between embryos (P0 vs. P1; P1 vs. P2) and using paired t -tests for comparisons between cytoplasmic and nuclear concentrations in either P1 or P2. In this and subsequent figures: * = P
    Figure Legend Snippet: Quantification of the increase in PIE-1::GFP concentration in the P lineage. A. Schematic of PIE-1 (gray) localization from the 1-cell to the 4-cell stage. Maternally deposited PIE-1 segregates asymmetrically to the germline blastomeres P1 and P2 during the first two rounds of cell division. PIE-1 is also degraded in somatic cells. Sister cells are connected by a line. B. Top panel: Coomassie stained SDS-PAGE gel of recombinant GFP and BSA, which was used as a loading standard. Bottom panels: Images of N2 and PIE-1::GFP embryos bathed in 300 nM GFP. Images were pseudocolored using the CyanHot lookup table in ImageJ (scale at the bottom). In order to highlight the dimmer PIE-1::GFP signals, the nuclear signal in the main image of the 4-cell embryo is saturated. The image normalization was adjusted equivalently in the 2 and 4-cell embryo insets such that the nuclear signal is not saturated. PIE-1::GFP concentration in the 1-cell embryo was determined using a 150 nM GFP bath, but is shown in a bath of 300 nM GFP to allow comparison with the later stage embryos. C. Estimates of PIE-1::GFP concentration in P0, P1 and P2. For P1 and P2, concentration estimates are shown for the entire cell (Tot), the cytoplasm (Cyt) and for the nucleus (Nuc). Mean concentrations and the number of embryos analyzed are indicated below the graph. Error bars represent 95% confidence intervals. Statistical significance was determined using unpaired t -tests with Welch’s correction for comparisons between embryos (P0 vs. P1; P1 vs. P2) and using paired t -tests for comparisons between cytoplasmic and nuclear concentrations in either P1 or P2. In this and subsequent figures: * = P

    Techniques Used: Concentration Assay, Staining, SDS Page, Recombinant

    7) Product Images from "Replication protein A promotes 5?- > 3? end processing during homology-dependent DNA double-strand break repair"

    Article Title: Replication protein A promotes 5?- > 3? end processing during homology-dependent DNA double-strand break repair

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201005110

    Functional and physical interactions between RPA and xDNA2. (A) The effect of RPA on xDNA2’s 5′→3′ exonuclease activity against two different single-stranded oligonucleotides. The substrates were labeled with 32 P-labeled dA (marked by the asterisks) and attached to Streptavidin paramagnetic beads via the 3′ biotin-dC. After incubation at room temperature for 1 h, the reactions were stopped with SDS-EDTA, boiled for 10 min, and separated on a 10% TAE-PAGE. The percentage of the substrate undegraded was relative to the total signal for each reaction. The sizes of the products were determined by separating on a sequencing gel (not depicted). (B) The effect of RPA and T4 gp32 on the nuclease activity of xDNA2. The substrate, 48mer-1 beads, was incubated with various proteins as indicated at room temperature for 1 h and analyzed similarly to that in A. (C) Coimmunoprecipitation of RPA and xDNA2. The immunoprecipitates were separated on an 8% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and probed for different proteins by Western blotting. For RPA, a rat antibody against the p70 subunit was used for Western blotting. Untreated cytosol was loaded at the indicated amounts to provide the standard for quantitation. White lines indicate that intervening lanes have been spliced out. (D) Interaction between the purified RPA and xDNA2. FLAG beads were precoated with either recombinant xDNA2 or BSA and then incubated with the purified RPA protein. The beads and supernatant fractions were analyzed similarly to that in C. xRPA, Xenopus RPA. Ab, antibody.
    Figure Legend Snippet: Functional and physical interactions between RPA and xDNA2. (A) The effect of RPA on xDNA2’s 5′→3′ exonuclease activity against two different single-stranded oligonucleotides. The substrates were labeled with 32 P-labeled dA (marked by the asterisks) and attached to Streptavidin paramagnetic beads via the 3′ biotin-dC. After incubation at room temperature for 1 h, the reactions were stopped with SDS-EDTA, boiled for 10 min, and separated on a 10% TAE-PAGE. The percentage of the substrate undegraded was relative to the total signal for each reaction. The sizes of the products were determined by separating on a sequencing gel (not depicted). (B) The effect of RPA and T4 gp32 on the nuclease activity of xDNA2. The substrate, 48mer-1 beads, was incubated with various proteins as indicated at room temperature for 1 h and analyzed similarly to that in A. (C) Coimmunoprecipitation of RPA and xDNA2. The immunoprecipitates were separated on an 8% SDS-PAGE, transferred to a polyvinylidene fluoride membrane, and probed for different proteins by Western blotting. For RPA, a rat antibody against the p70 subunit was used for Western blotting. Untreated cytosol was loaded at the indicated amounts to provide the standard for quantitation. White lines indicate that intervening lanes have been spliced out. (D) Interaction between the purified RPA and xDNA2. FLAG beads were precoated with either recombinant xDNA2 or BSA and then incubated with the purified RPA protein. The beads and supernatant fractions were analyzed similarly to that in C. xRPA, Xenopus RPA. Ab, antibody.

    Techniques Used: Functional Assay, Recombinase Polymerase Amplification, Activity Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Sequencing, SDS Page, Western Blot, Quantitation Assay, Purification, Recombinant

    8) Product Images from "The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1"

    Article Title: The histone H3K36 demethylase Rph1/KDM4 regulates the expression of the photoreactivation gene PHR1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr040

    The phospho-mutant at S652 of Rph1 increases UV sensitivity and impairs the dissociation after UV irradiation. ( A ) In vitro kinase assay was performed by recombinant Rph1 or BSA incubated with or without V5-IP WT or KD Rad53 supplied by γ 32 P-ATP. The signal was detected by autoradiography. pRad53 indicated the autophosphorylation of Rad53. pRph1 indicated the phosphorylation of Rph1. Coomassie Blue and immnoblotting (anti-V5) showed the loading controls. ( B ) UV sensitivity of rph1Δ cells containing control vector, WT Rph1 ( RPH1 ) or phospho-defective Rph1 mutants. ( C and D ) The indicated strains as in (B) were harvested for RT-qPCR to detect PHR1 expression in response to UV or not (C) and for HA-ChIP to measure the association of Rph1 at URS of PHR1 (D). Error bars show the SD of three biological repeats. * P
    Figure Legend Snippet: The phospho-mutant at S652 of Rph1 increases UV sensitivity and impairs the dissociation after UV irradiation. ( A ) In vitro kinase assay was performed by recombinant Rph1 or BSA incubated with or without V5-IP WT or KD Rad53 supplied by γ 32 P-ATP. The signal was detected by autoradiography. pRad53 indicated the autophosphorylation of Rad53. pRph1 indicated the phosphorylation of Rph1. Coomassie Blue and immnoblotting (anti-V5) showed the loading controls. ( B ) UV sensitivity of rph1Δ cells containing control vector, WT Rph1 ( RPH1 ) or phospho-defective Rph1 mutants. ( C and D ) The indicated strains as in (B) were harvested for RT-qPCR to detect PHR1 expression in response to UV or not (C) and for HA-ChIP to measure the association of Rph1 at URS of PHR1 (D). Error bars show the SD of three biological repeats. * P

    Techniques Used: Mutagenesis, Irradiation, In Vitro, Kinase Assay, Recombinant, Incubation, Autoradiography, Plasmid Preparation, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation

    9) Product Images from "Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion"

    Article Title: Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2011.07927.x

    Screening of EHEC O157:H7 O-island (OI) mutants for altered levels of T3S. SDS-PAGE gels showing culture supernatant secretion profiles for EHEC strain TUV93-0 and a selection of 24 isogenic deletion strains. Parent strain TUV93-0 also acted a positive control for T3S and a T3S system mutant (ΔLEE1–3) provided a negative control for secretion. OI deletions are as defined with reference to the original designations in Perna et al . (2001 ). The translocon protein bands are indicated, EspB/D and EspA as well as BSA which was added as a loading control to rule out the precipitation process as a source of variation and to act as a co-precipitant. Strains were cultured in MEM-HEPES to an OD 600 of 1.0 and culture supernatants were TCA-precipitated, separated by SDS-PAGE and stained with Colloidal blue as described in Experimental procedures .
    Figure Legend Snippet: Screening of EHEC O157:H7 O-island (OI) mutants for altered levels of T3S. SDS-PAGE gels showing culture supernatant secretion profiles for EHEC strain TUV93-0 and a selection of 24 isogenic deletion strains. Parent strain TUV93-0 also acted a positive control for T3S and a T3S system mutant (ΔLEE1–3) provided a negative control for secretion. OI deletions are as defined with reference to the original designations in Perna et al . (2001 ). The translocon protein bands are indicated, EspB/D and EspA as well as BSA which was added as a loading control to rule out the precipitation process as a source of variation and to act as a co-precipitant. Strains were cultured in MEM-HEPES to an OD 600 of 1.0 and culture supernatants were TCA-precipitated, separated by SDS-PAGE and stained with Colloidal blue as described in Experimental procedures .

    Techniques Used: SDS Page, Selection, Positive Control, Mutagenesis, Negative Control, Activated Clotting Time Assay, Cell Culture, Staining

    10) Product Images from "Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks"

    Article Title: Human RECQ1 Interacts with Ku70/80 and Modulates DNA End-Joining of Double-Strand Breaks

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0062481

    A direct physical interaction between RECQ1 and Ku70/80 in vitro . A. RECQ1 directly interacts with Ku70 and Ku80. GST or GST fused-full length RECQ1 was incubated with bacterially expressed Ku70/80, Ku70, Ku80 or N-Ku80 (lacking the C-terminus amino acid residues 565–732) followed by extensive washing of the beads, SDS-PAGE, and Western transfer. The blots were probed separately with anti-His (for Ku detection) and anti-RECQ1 antibodies. Input lanes account for 10% of the bacterial lysate expressing Ku protein used in the pull-down reactions. B. Recombinant RECQ1 and Ku70/80 proteins interact directly as shown by ELISA. Either BSA or purified recombinant Ku70/80 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant RECQ1 (0–50 nM) for 1 h at 30°C. Parallel wells contained DNaseI (100 U/ml) or EtBr (50 µg/ml) in the binding step to test for DNA-mediated protein interaction. Following washing, Ku70/80-bound RECQ1 was detected by ELISA using anti-RECQ1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. C. GST alone or GST-RECQ1 fragments (as indicated) bound to glutathione beads were incubated overnight at 4°C with HeLa extract (500 µg) that was either untreated or pre-treated with benzonase. After extensive washings, the bound Ku70/80 was eluted with SDS sample buffer and analyzed by Western blot using anti-Ku70 and anti-Ku80 antibodies (right). Coomassie staining of the eluted proteins was done to test expression of various GST-fusion fragments of RECQ1 (left). GST-RECQ1 proteins are marked by asterisk. Marker, protein molecular weight marker.
    Figure Legend Snippet: A direct physical interaction between RECQ1 and Ku70/80 in vitro . A. RECQ1 directly interacts with Ku70 and Ku80. GST or GST fused-full length RECQ1 was incubated with bacterially expressed Ku70/80, Ku70, Ku80 or N-Ku80 (lacking the C-terminus amino acid residues 565–732) followed by extensive washing of the beads, SDS-PAGE, and Western transfer. The blots were probed separately with anti-His (for Ku detection) and anti-RECQ1 antibodies. Input lanes account for 10% of the bacterial lysate expressing Ku protein used in the pull-down reactions. B. Recombinant RECQ1 and Ku70/80 proteins interact directly as shown by ELISA. Either BSA or purified recombinant Ku70/80 was coated onto microtiter plates. Following blocking with 3% BSA, appropriate wells were incubated with the indicated concentrations of recombinant RECQ1 (0–50 nM) for 1 h at 30°C. Parallel wells contained DNaseI (100 U/ml) or EtBr (50 µg/ml) in the binding step to test for DNA-mediated protein interaction. Following washing, Ku70/80-bound RECQ1 was detected by ELISA using anti-RECQ1 antibody. The values represent the mean of three independent experiments performed in duplicate with SD indicated by error bars. C. GST alone or GST-RECQ1 fragments (as indicated) bound to glutathione beads were incubated overnight at 4°C with HeLa extract (500 µg) that was either untreated or pre-treated with benzonase. After extensive washings, the bound Ku70/80 was eluted with SDS sample buffer and analyzed by Western blot using anti-Ku70 and anti-Ku80 antibodies (right). Coomassie staining of the eluted proteins was done to test expression of various GST-fusion fragments of RECQ1 (left). GST-RECQ1 proteins are marked by asterisk. Marker, protein molecular weight marker.

    Techniques Used: In Vitro, Incubation, SDS Page, Western Blot, Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Purification, Blocking Assay, Binding Assay, Staining, Marker, Molecular Weight

    11) Product Images from "Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase"

    Article Title: Dimerization and opposite base-dependent catalytic impairment of polymorphic S326C OGG1 glycosylase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl060

    Size-exclusion chromatographic analysis of wild-type and S326C OGG1. ( A ) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min ( B ). Identical runs were performed with 100 µg polymorphic S326C OGG1 ( C ) or 100 µg of both wild-type and S326C OGG1 together ( D ).
    Figure Legend Snippet: Size-exclusion chromatographic analysis of wild-type and S326C OGG1. ( A ) Non-denatured protein size markers (Sigma). Peak 1, BSA dimer (132 kDa); peak 2, BSA monomer (66 kDa) and peak 3, carbonic anydrase (29 kDa). Purified wild-type OGG1 (100 µg) was analyzed on a Superdex 200 HR column equilibrated with 20 mM Tris–HCl (pH 7.4), 300 mM NaCl at a flow rate of 0.25 ml/min ( B ). Identical runs were performed with 100 µg polymorphic S326C OGG1 ( C ) or 100 µg of both wild-type and S326C OGG1 together ( D ).

    Techniques Used: Purification, Flow Cytometry

    12) Product Images from "RNA Chaperone Function of a Universal Stress Protein in Arabidopsis Confers Enhanced Cold Stress Tolerance in Plants"

    Article Title: RNA Chaperone Function of a Universal Stress Protein in Arabidopsis Confers Enhanced Cold Stress Tolerance in Plants

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18122546

    Nucleic acid-binding activity of AtUSP in vitro. Indicated amounts of purified recombinant AtUSP protein were incubated with either ( A ) M13mp8 ssDNA, ( B ) M13mp8 dsDNA, or ( C ) in vitro transcribed luciferase ( luc ) mRNA. To analyze the effect of AtUSP in RNA mobility and the AtUSP-RNA complexes, 0.8% agarose gels were used for gel-shift assays. Bovine serum albumin (BSA) protein (100 μg/μL) was used as a negative control. SM presents size marker from Thermo Scientific Company.
    Figure Legend Snippet: Nucleic acid-binding activity of AtUSP in vitro. Indicated amounts of purified recombinant AtUSP protein were incubated with either ( A ) M13mp8 ssDNA, ( B ) M13mp8 dsDNA, or ( C ) in vitro transcribed luciferase ( luc ) mRNA. To analyze the effect of AtUSP in RNA mobility and the AtUSP-RNA complexes, 0.8% agarose gels were used for gel-shift assays. Bovine serum albumin (BSA) protein (100 μg/μL) was used as a negative control. SM presents size marker from Thermo Scientific Company.

    Techniques Used: Binding Assay, Activity Assay, In Vitro, Purification, Recombinant, Incubation, Luciferase, Electrophoretic Mobility Shift Assay, Negative Control, Marker

    13) Product Images from "High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection"

    Article Title: High-Throughput Protein Expression Using a Combination of Ligation-Independent Cloning (LIC) and Infrared Fluorescent Protein (IFP) Detection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018900

    Protein-DNA interaction analysis based on IFP fusions. ( A ) Fusion proteins ANAC042-TEV-IFP-6xHis (70 kDa) and IFP-6xHis (36 kDa) affinity-purified from E. coli . Two elution fractions (2×250 µL) containing the purified proteins were pooled and analysed after SDS-PAGE by in-gel detection (top) and Coomassie staining (bottom) (lanes 10–13: ANAC042-TEV-IFP-6xHis, 5, 10, 15, and 20 µL; lanes 6 - 9: IFP-6xHis, 2, 5, 8 and 10 µL). BSA served as standard to estimate protein amounts (lanes 1–5: 100/250/500/750/1000 ng). Equal amounts of both proteins (∼5 µg) were used for protein-DNA interaction analysis. M, molecular mass marker (kDa). ( B ) Biotinylated dsDNA was immobilized on streptavidin mutein particles and incubated with ANAC042-TEV-IFP-6xHis protein. After elution, fractions were scanned at 700 nm in the wells of a microtiter plate (strong infrared signal appears white in the digital image). A1: IFP-6xHis input. A2: ANAC042-TEV-IFP-6xHis input. A3: negative control; B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis in the presence of non-biotinylated 7%-DNA. A4: negative control; B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. B1/2: empty wells. B3/4: experiments with B-100%-DNA and B-7%-DNA immobilized on streptavidin mutein beads + ANAC042-TEV-IFP-6xHis incubated in the presence of non-biotinylated 7%- and 100%-DNA, respectively. Areas of the infrared signals were marked (white circles) and integrated signal intensities were calculated (B3 = 319, and B4 = 50). ( C ) After infrared-scanning in microtiter plates (see B) samples were separated by SDS-PAGE and scanned at 700 nm (top) followed by western blot analysis (bottom). Lane 1: IFP-6xHis input (white square). Lane 2: ANAC042-TEV-IFP-6xHis input (white square). Lane 3: negative control with B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 4: experiment with B-100%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 5: negative control with B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. Lane 6: experiment with B-7%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 100%-DNA.
    Figure Legend Snippet: Protein-DNA interaction analysis based on IFP fusions. ( A ) Fusion proteins ANAC042-TEV-IFP-6xHis (70 kDa) and IFP-6xHis (36 kDa) affinity-purified from E. coli . Two elution fractions (2×250 µL) containing the purified proteins were pooled and analysed after SDS-PAGE by in-gel detection (top) and Coomassie staining (bottom) (lanes 10–13: ANAC042-TEV-IFP-6xHis, 5, 10, 15, and 20 µL; lanes 6 - 9: IFP-6xHis, 2, 5, 8 and 10 µL). BSA served as standard to estimate protein amounts (lanes 1–5: 100/250/500/750/1000 ng). Equal amounts of both proteins (∼5 µg) were used for protein-DNA interaction analysis. M, molecular mass marker (kDa). ( B ) Biotinylated dsDNA was immobilized on streptavidin mutein particles and incubated with ANAC042-TEV-IFP-6xHis protein. After elution, fractions were scanned at 700 nm in the wells of a microtiter plate (strong infrared signal appears white in the digital image). A1: IFP-6xHis input. A2: ANAC042-TEV-IFP-6xHis input. A3: negative control; B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis in the presence of non-biotinylated 7%-DNA. A4: negative control; B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. B1/2: empty wells. B3/4: experiments with B-100%-DNA and B-7%-DNA immobilized on streptavidin mutein beads + ANAC042-TEV-IFP-6xHis incubated in the presence of non-biotinylated 7%- and 100%-DNA, respectively. Areas of the infrared signals were marked (white circles) and integrated signal intensities were calculated (B3 = 319, and B4 = 50). ( C ) After infrared-scanning in microtiter plates (see B) samples were separated by SDS-PAGE and scanned at 700 nm (top) followed by western blot analysis (bottom). Lane 1: IFP-6xHis input (white square). Lane 2: ANAC042-TEV-IFP-6xHis input (white square). Lane 3: negative control with B-100%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 4: experiment with B-100%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 7%-DNA. Lane 5: negative control with B-7%-DNA immobilized on streptavidin mutein particles + IFP-6xHis, in the presence of non-biotinylated 100%-DNA. Lane 6: experiment with B-7%-DNA immobilized on streptavidin mutein particles + ANAC042-TEV-IFP-6xHis, in the presence of non-biotinylated 100%-DNA.

    Techniques Used: Affinity Purification, Purification, SDS Page, Staining, Marker, Incubation, Negative Control, Western Blot

    14) Product Images from "Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System"

    Article Title: Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00699

    Diagram of the modified Gln-tRNA-gRNA cassettes in pUC19-tRp-gRNA and pCas9-tRp-gRNA vectors. The blue arrow on the left is the Gln-tRNA promoter that is followed by the gRNA spacer insert site, gRNA scaffold (green), and RNA polyIII terminator (purple) sequences. The gRNA spacers are synthesized by annealing the sense and antisense oligonucleotides with 5′-ACCT and 3′-CAAA overhangs and inserted into the gRNA spacer insertion site of Bsa I-digested pUC19-tRp-gRNA (upper) or Bsm BI-digested pCas9-tRp-gRNA (lower). The gRNA start site is marked with a red arrow. The cleavage sites of type II restriction enzyme Bsa I and Bsm BI are marked with black arrows.
    Figure Legend Snippet: Diagram of the modified Gln-tRNA-gRNA cassettes in pUC19-tRp-gRNA and pCas9-tRp-gRNA vectors. The blue arrow on the left is the Gln-tRNA promoter that is followed by the gRNA spacer insert site, gRNA scaffold (green), and RNA polyIII terminator (purple) sequences. The gRNA spacers are synthesized by annealing the sense and antisense oligonucleotides with 5′-ACCT and 3′-CAAA overhangs and inserted into the gRNA spacer insertion site of Bsa I-digested pUC19-tRp-gRNA (upper) or Bsm BI-digested pCas9-tRp-gRNA (lower). The gRNA start site is marked with a red arrow. The cleavage sites of type II restriction enzyme Bsa I and Bsm BI are marked with black arrows.

    Techniques Used: Modification, Synthesized

    15) Product Images from "Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth ▿"

    Article Title: Legionella pneumophilaRequires Polyamines for Optimal Intracellular Growth ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01506-10

    HtpB interacts with mammalian SAMDC. (a) Far-Western blot assay with 5 or 10 μg of immobilized purified proteins (HtpB, GroEL, and BSA) overlaid with a lysate of L929 cells (L). The negative control for the SAMDC immunostaining signal was prepared
    Figure Legend Snippet: HtpB interacts with mammalian SAMDC. (a) Far-Western blot assay with 5 or 10 μg of immobilized purified proteins (HtpB, GroEL, and BSA) overlaid with a lysate of L929 cells (L). The negative control for the SAMDC immunostaining signal was prepared

    Techniques Used: Far Western Blot, Purification, Negative Control, Immunostaining

    16) Product Images from "The Ska complex promotes Aurora B activity to ensure chromosome biorientation"

    Article Title: The Ska complex promotes Aurora B activity to ensure chromosome biorientation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201603019

    The Ska complex promotes the catalytic activity of Aurora B in vitro. (A) Time course kinase assay with Aurora B–His and MBP–INCENP 790–919 –His preincubated with Ska complex or equimolar amounts of BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (B) Quantification of histone H3 32 P signals from A. Signals were normalized to H3 and Aurora B protein levels monitored by Ponceau S staining (Ponc. S) and Western blotting (WB), respectively. Signal intensities are expressed relative to the first time-point. Data represent mean ± SD (three experiments). (C) Aurora B–His was incubated with recombinant Ska complex before pull-down with beads coupled to anti-Ska1 antibody or control antibody (IgG). UB, unbound fraction; B, bound fraction. (D) Immunoprecipitates (IP) from mitotic HeLa S3 cell extracts, obtained using anti-Ska1 antibodies or control antibodies (IgG), analyzed by WB. (E) Aurora B–His was preincubated with Ska complex (comp.) or histone H3, as control, before incubation with γ-[ 32 P]ATP. Aurora B autophosphorylation is visualized by autoradiography ( 32 P) and Aurora B levels by Coomassie Brilliant Blue (CBB) staining (see Fig. S5 G for uncropped results). (F) Quantification of Aurora B autophosphorylation signals from E (one experiment). Signal intensities are expressed relative to the first concentration. (G) Time course kinase assay with recombinant Aurora B–His preincubated with Ska complex, equimolar amounts of MBP–INCENP 790–919 –His or BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (H) Quantification of Aurora B kinase activity as in B (one experiment).
    Figure Legend Snippet: The Ska complex promotes the catalytic activity of Aurora B in vitro. (A) Time course kinase assay with Aurora B–His and MBP–INCENP 790–919 –His preincubated with Ska complex or equimolar amounts of BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (B) Quantification of histone H3 32 P signals from A. Signals were normalized to H3 and Aurora B protein levels monitored by Ponceau S staining (Ponc. S) and Western blotting (WB), respectively. Signal intensities are expressed relative to the first time-point. Data represent mean ± SD (three experiments). (C) Aurora B–His was incubated with recombinant Ska complex before pull-down with beads coupled to anti-Ska1 antibody or control antibody (IgG). UB, unbound fraction; B, bound fraction. (D) Immunoprecipitates (IP) from mitotic HeLa S3 cell extracts, obtained using anti-Ska1 antibodies or control antibodies (IgG), analyzed by WB. (E) Aurora B–His was preincubated with Ska complex (comp.) or histone H3, as control, before incubation with γ-[ 32 P]ATP. Aurora B autophosphorylation is visualized by autoradiography ( 32 P) and Aurora B levels by Coomassie Brilliant Blue (CBB) staining (see Fig. S5 G for uncropped results). (F) Quantification of Aurora B autophosphorylation signals from E (one experiment). Signal intensities are expressed relative to the first concentration. (G) Time course kinase assay with recombinant Aurora B–His preincubated with Ska complex, equimolar amounts of MBP–INCENP 790–919 –His or BSA, as control, before addition of histone H3 and γ-[ 32 P]ATP. (H) Quantification of Aurora B kinase activity as in B (one experiment).

    Techniques Used: Activity Assay, In Vitro, Kinase Assay, Staining, Western Blot, Incubation, Recombinant, Autoradiography, Concentration Assay

    17) Product Images from "Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA"

    Article Title: Dimerization and DNA-dependent aggregation of the Escherichia coli nucleoid protein and chaperone CbpA

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2010.07292.x

    CbpA protects plasmid DNA from degradation by nucleases. A. The panel shows naked plasmid and complexes with CbpA run on a 1% agarose gel. Plasmid (30 ng) was pre-incubated with 0, 0.5, 1.0, or 2.0 µM CbpA. Note that only every other lane has been loaded on the gel. B. The panel shows plasmid run on a 1% agarose gel. Plasmids (77 ng) were treated with different combinations of DNase I, BSA (1, 2 or 3 µM) and CbpA (1, 2 or 3 µM).
    Figure Legend Snippet: CbpA protects plasmid DNA from degradation by nucleases. A. The panel shows naked plasmid and complexes with CbpA run on a 1% agarose gel. Plasmid (30 ng) was pre-incubated with 0, 0.5, 1.0, or 2.0 µM CbpA. Note that only every other lane has been loaded on the gel. B. The panel shows plasmid run on a 1% agarose gel. Plasmids (77 ng) were treated with different combinations of DNase I, BSA (1, 2 or 3 µM) and CbpA (1, 2 or 3 µM).

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

    18) Product Images from "Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System"

    Article Title: Targeted Deletion of the USTA and UvSLT2 Genes Efficiently in Ustilaginoidea virens With the CRISPR-Cas9 System

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2018.00699

    Diagram of the modified Gln-tRNA-gRNA cassettes in pUC19-tRp-gRNA and pCas9-tRp-gRNA vectors. The blue arrow on the left is the Gln-tRNA promoter that is followed by the gRNA spacer insert site, gRNA scaffold (green), and RNA polyIII terminator (purple) sequences. The gRNA spacers are synthesized by annealing the sense and antisense oligonucleotides with 5′-ACCT and 3′-CAAA overhangs and inserted into the gRNA spacer insertion site of Bsa I-digested pUC19-tRp-gRNA (upper) or Bsm BI-digested pCas9-tRp-gRNA (lower). The gRNA start site is marked with a red arrow. The cleavage sites of type II restriction enzyme Bsa I and Bsm BI are marked with black arrows.
    Figure Legend Snippet: Diagram of the modified Gln-tRNA-gRNA cassettes in pUC19-tRp-gRNA and pCas9-tRp-gRNA vectors. The blue arrow on the left is the Gln-tRNA promoter that is followed by the gRNA spacer insert site, gRNA scaffold (green), and RNA polyIII terminator (purple) sequences. The gRNA spacers are synthesized by annealing the sense and antisense oligonucleotides with 5′-ACCT and 3′-CAAA overhangs and inserted into the gRNA spacer insertion site of Bsa I-digested pUC19-tRp-gRNA (upper) or Bsm BI-digested pCas9-tRp-gRNA (lower). The gRNA start site is marked with a red arrow. The cleavage sites of type II restriction enzyme Bsa I and Bsm BI are marked with black arrows.

    Techniques Used: Modification, Synthesized

    19) Product Images from "Electrochemical Assay for the Signal-on Detection of Human DNA Methyltransferase Activity"

    Article Title: Electrochemical Assay for the Signal-on Detection of Human DNA Methyltransferase Activity

    Journal: Journal of the American Chemical Society

    doi: 10.1021/ja4085918

    Protease treatment restores peak sharpness for samples of high protein content. Chips were modified in all quadrants with the hemimethylated BssH II 22-mer. One half of the chip was treated with 100 nM Dnmt1 with 100 μg/mL BSA and 160 μM
    Figure Legend Snippet: Protease treatment restores peak sharpness for samples of high protein content. Chips were modified in all quadrants with the hemimethylated BssH II 22-mer. One half of the chip was treated with 100 nM Dnmt1 with 100 μg/mL BSA and 160 μM

    Techniques Used: Modification, Chromatin Immunoprecipitation

    20) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Mobius Assembly standard part generation. (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.
    Figure Legend Snippet: Mobius Assembly standard part generation. (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.

    Techniques Used: Plasmid Preparation, Clone Assay, Marker, Amplification, Functional Assay, Sequencing

    21) Product Images from "Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly"

    Article Title: Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0189892

    Mobius Assembly standard part generation. (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.
    Figure Legend Snippet: Mobius Assembly standard part generation. (A) Mobius Universal Acceptor Vector (mUAV) is the vector which converts and hosts DNA fragments as standard parts. mUAV is flanked by the Type IIS restriction enzymes Bsa I and Aar I and carries amilCP gene as visible cloning screening marker. The inserts are amplified with primers containing Aar I recognition sites, the fusion sites with the mUAV, and the standard overhangs, and they replace amilCP cassette in a Golden Gate reaction. The standard parts are released by Bsa I digestion. E: Eco RI; P: Pst I. (B) Mobius Assembly embraces the 4bp standard part overhangs defined by MoClo, Golden Braid, and Phytobricks, to facilitate part sharing. The middle row illustrates the standard overhangs for major functional parts (promoter, coding sequence, and terminator); the top row shows the recommended overhangs for eukaryotic sub-functional parts, while the bottom row indicates ones for the prokaryotic counterparts.

    Techniques Used: Plasmid Preparation, Clone Assay, Marker, Amplification, Functional Assay, Sequencing

    22) Product Images from "Repair of Gaps in Retroviral DNA Integration Intermediates"

    Article Title: Repair of Gaps in Retroviral DNA Integration Intermediates

    Journal: Journal of Virology

    doi:

    Blocking of polymerase access to DNA gaps by added integrase. (A) A four-arm substrate was synthesized with two arms matching RSV cDNA sequences (bold lines) and two arms of mixed sequence recapitulating target DNA (thin lines). Two separate molecules with complementary gap sequences were studied to permit assembly of the four-armed structure. Addition of polymerase and a labeled nucleotide (asterisks) results in incorporation of radioactive nucleotides in the gap. Addition of integrase, in contrast, can potentially block access. (B) Polymerization on the RSV gapped dumbbell substrate. Labeled DNA products indicates incorporation of 32 P-labeled dCTPs in the dumbbell substrate by Pol beta. RSV integrase was preincubated for 10 min with the substrate in increasing amounts; lanes 2 to 6 contained 0.1, 1, 10, 50, and 100 ng, respectively. BSA was similarly preincubated with the substrate in lanes 7 to 11 in increasing amounts (0.1, 1, 10, 50, and 100 ng, respectively). The preincubation was followed by the addition of Pol beta and further incubation for 10 min. The amounts of relative incorporation in lanes 2 to 6, respectively, were 100, 108, 34, 15, and 2%. The amounts of relative incorporation in lanes 7 to 11, respectively, were 100, 107, 91, 321, and 108%. (C) Polymerization on a 5′-labeled nicked substrate. The diagrams at the left indicate the unreacted nicked substrate and a strand displacement synthesis product. Reaction compositions were the same as for panel B except for the DNA substrate. The relative percent conversion values for lanes 2 to 6 were, respectively, 100, 113, 115, 87, and 58%. The relative percent conversion values for lanes 7 to 11 were, respectively, 100, 88, 152, 127, and 126%.
    Figure Legend Snippet: Blocking of polymerase access to DNA gaps by added integrase. (A) A four-arm substrate was synthesized with two arms matching RSV cDNA sequences (bold lines) and two arms of mixed sequence recapitulating target DNA (thin lines). Two separate molecules with complementary gap sequences were studied to permit assembly of the four-armed structure. Addition of polymerase and a labeled nucleotide (asterisks) results in incorporation of radioactive nucleotides in the gap. Addition of integrase, in contrast, can potentially block access. (B) Polymerization on the RSV gapped dumbbell substrate. Labeled DNA products indicates incorporation of 32 P-labeled dCTPs in the dumbbell substrate by Pol beta. RSV integrase was preincubated for 10 min with the substrate in increasing amounts; lanes 2 to 6 contained 0.1, 1, 10, 50, and 100 ng, respectively. BSA was similarly preincubated with the substrate in lanes 7 to 11 in increasing amounts (0.1, 1, 10, 50, and 100 ng, respectively). The preincubation was followed by the addition of Pol beta and further incubation for 10 min. The amounts of relative incorporation in lanes 2 to 6, respectively, were 100, 108, 34, 15, and 2%. The amounts of relative incorporation in lanes 7 to 11, respectively, were 100, 107, 91, 321, and 108%. (C) Polymerization on a 5′-labeled nicked substrate. The diagrams at the left indicate the unreacted nicked substrate and a strand displacement synthesis product. Reaction compositions were the same as for panel B except for the DNA substrate. The relative percent conversion values for lanes 2 to 6 were, respectively, 100, 113, 115, 87, and 58%. The relative percent conversion values for lanes 7 to 11 were, respectively, 100, 88, 152, 127, and 126%.

    Techniques Used: Blocking Assay, Synthesized, Sequencing, Labeling, Incubation

    23) Product Images from "Structural and Functional Characterization of IS679 and IS66-Family Elements"

    Article Title: Structural and Functional Characterization of IS679 and IS66-Family Elements

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.183.14.4296-4304.2001

    (A) Schematic representation of the IS 679 structure. IS 679 (2,704 bp) has imperfect 25-bp IRs. The IRs at the left and right inverted repeats (IRL and IRR) are indicated by solid triangles. Open, dotted, and cross-hatched arrows indicate, respectively, tnpA, tnpB , and tnpC . The two cross-hatched ovals flanking IS 679 indicate direct repeats of an 8-bp target site sequence. (B) Schematic representations of the structures of pHAN plasmids. pHAN103 carries Tn 679 with the kanamycin resistance gene (Km r ) between an intact IS 679 sequence and the 3′-end region having IRR. Plasmids pHAN104, pHAN105, and pHAN106 carry a Tn 679 derivative with deletions (hatched box) in tnpA, tnpB , and tnpC (thin arrows), respectively. Small solid arrows beneath the pHAN plasmid indicate primers used to construct each plasmid (see Materials and Methods). Primers with a tail indicate an additional sequence with a restriction site. s, Sac II; ai, Bsa I; ei, Bsp EI; gi, Bsr GI; r, Rsr II.
    Figure Legend Snippet: (A) Schematic representation of the IS 679 structure. IS 679 (2,704 bp) has imperfect 25-bp IRs. The IRs at the left and right inverted repeats (IRL and IRR) are indicated by solid triangles. Open, dotted, and cross-hatched arrows indicate, respectively, tnpA, tnpB , and tnpC . The two cross-hatched ovals flanking IS 679 indicate direct repeats of an 8-bp target site sequence. (B) Schematic representations of the structures of pHAN plasmids. pHAN103 carries Tn 679 with the kanamycin resistance gene (Km r ) between an intact IS 679 sequence and the 3′-end region having IRR. Plasmids pHAN104, pHAN105, and pHAN106 carry a Tn 679 derivative with deletions (hatched box) in tnpA, tnpB , and tnpC (thin arrows), respectively. Small solid arrows beneath the pHAN plasmid indicate primers used to construct each plasmid (see Materials and Methods). Primers with a tail indicate an additional sequence with a restriction site. s, Sac II; ai, Bsa I; ei, Bsp EI; gi, Bsr GI; r, Rsr II.

    Techniques Used: Sequencing, Plasmid Preparation, Construct

    Related Articles

    Diagnostic Assay:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes) .. Pipettes (P2, P10, P20, P100, P200) Table-top heat block (Digital Dry Block Heater, VWR) Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2) Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500)

    Centrifugation:

    Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation
    Article Snippet: Diluted chromatin samples were pre-cleared for 1 h with 55 μL of 50 % suspension of protein A sepharose (Protein A Sepharose CL-4B, GE Healthcare N: 17-0780-01) beads that were blocked with 0.2 μg/μL sonicated herring sperm DNA (Thermo Fisher Scientific, N: 15634-017) and 0.5 μg/μL BSA (B9000 S, NEB). .. Immune complexes were collected with 75 μL of 50 % suspension of protein A Sepharose for 1 h at 4 °C followed by centrifugation at 1000 rpm and 4 °C for 5 min using a table top centrifuge.

    Luciferase:

    Article Title: A retained intron in the 3′‐ UTR of Calm3 mRNA mediates its Staufen2‐ and activity‐dependent localization to neuronal dendrites
    Article Snippet: For detecting mRNA levels by qPCR, a homemade SYBR Green Mix [containing the following components at a final concentration of 1 M betaine (B0300; Sigma), 1× standard Taq buffer (NEB), 16 μM dNTPs (N0447S; NEB), BSA 20 μg/ml (B9000S; NEB), 0.6 U per reaction Hot‐Start Taq DNA polymerase (M0495S; NEB), and 1 μl/ml of 1:100 SYBR Green (20010; Lumiprobe)] (in ddH2 O) was used. .. Forward and reverse primer pair mix was used 2 μl per reaction from the following stock concentrations: Renilla and firefly luciferase at 3 μM, GFP 5 μM, and Calm3 ORF Fwd/Rev 2.5 μM; Calm3 ORF Fwd/Calm3 intron Rev 4 μM, Stau2 5 μM, pp1a 3 μM, GFP Fwd/Calm3 intron Rev 4 μM, and Renilla luciferase Fwd/Calm3 intron Rev 4 μM.

    Quantitative RT-PCR:

    Article Title: A retained intron in the 3′‐ UTR of Calm3 mRNA mediates its Staufen2‐ and activity‐dependent localization to neuronal dendrites
    Article Snippet: Paragraph title: cDNA synthesis and quantitative RT–PCR ... For detecting mRNA levels by qPCR, a homemade SYBR Green Mix [containing the following components at a final concentration of 1 M betaine (B0300; Sigma), 1× standard Taq buffer (NEB), 16 μM dNTPs (N0447S; NEB), BSA 20 μg/ml (B9000S; NEB), 0.6 U per reaction Hot‐Start Taq DNA polymerase (M0495S; NEB), and 1 μl/ml of 1:100 SYBR Green (20010; Lumiprobe)] (in ddH2 O) was used.

    Real-time Polymerase Chain Reaction:

    Article Title: A retained intron in the 3′‐ UTR of Calm3 mRNA mediates its Staufen2‐ and activity‐dependent localization to neuronal dendrites
    Article Snippet: .. For detecting mRNA levels by qPCR, a homemade SYBR Green Mix [containing the following components at a final concentration of 1 M betaine (B0300; Sigma), 1× standard Taq buffer (NEB), 16 μM dNTPs (N0447S; NEB), BSA 20 μg/ml (B9000S; NEB), 0.6 U per reaction Hot‐Start Taq DNA polymerase (M0495S; NEB), and 1 μl/ml of 1:100 SYBR Green (20010; Lumiprobe)] (in ddH2 O) was used. .. Forward and reverse primer pair mix was used 2 μl per reaction from the following stock concentrations: Renilla and firefly luciferase at 3 μM, GFP 5 μM, and Calm3 ORF Fwd/Rev 2.5 μM; Calm3 ORF Fwd/Calm3 intron Rev 4 μM, Stau2 5 μM, pp1a 3 μM, GFP Fwd/Calm3 intron Rev 4 μM, and Renilla luciferase Fwd/Calm3 intron Rev 4 μM.

    Incubation:

    Article Title: RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2
    Article Snippet: .. RNA was incubated for 2 min at 95 °C in 10 mM Tris-HCl pH 7.5 (at 25 °C) and was then immediately snap-cooled on ice for 2 min. Next, RNA was allowed to fold for 30 min at 37 °C in binding buffer (50 mM Tris-HCl pH 7.5 at 25 °C, 200 mM KCl, 2.5 mM MgCl2 , 0.1 mM ZnCl2 , 2 mM 2-mercaptoethanol, 0.1 mg/mL bovine serum albumin (NEB #B9000S), 0.05% Nonidet P40 (Roche #11754599001) and 0.1 mg/mL fragmented yeast tRNA (Sigma #R5636)). .. For assaying the RNA-binding affinity in the presence or absence of stimulatory or substrate peptides, binding buffer was adjusted to mimic conditions used for HMTase assays with these peptides and consisted of 50 mM Tris-HCl pH 7.5 (at 25 °C), 100 mM KCl, 2 mM 2-mercaptoethanol, 0.05% Nonidet P40 (Roche #11754599001) and 0.1 mg/mL bovine serum albumin (NEB #B9000S).

    Article Title: RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples
    Article Snippet: .. Finally, samples were incubated in RCA buffer containing 1U/μl phi29 polymerase (Thermo, cat. no. EP0091), 1 × phi29 polymerase buffer, 0.25 mM dNTPs (Thermo, cat. no. R0191), 0.2 μg/μl BSA (NEB cat. no. B9000S) and 5% glycerol in DEPC-H2 O for 1 h or otherwise specified. .. Samples were then washed twice in DEPC-PBS-T, and incubated in a buffer containing 2 × SSC, 20% formamide, and 100 nM of the detection oligos at 37 °C for 30 min. Unbound detection oligos were removed with two DEPC-PBS-T washes.

    Article Title: Spatial detection of fetal marker genes expressed at low level in adult human heart tissue
    Article Snippet: .. On-chip permeabilization, reverse transcription and probe-release reactions Tissue sections on oligonucleotide-covered glass slides were placed in ArrayIT mask holders to create reaction chambers for each tissue section, and incubated in 70 μl of a pre-permeabilization mixture [1x ExoI buffer (#B0293S, NEB), 0.2 μg/μl BSA (#B9000S, NEB)] at 37 °C for 30 minutes then washed in 100 μl 0.1x SSC (#S6639, Sigma-Aldrich) diluted in MQ RNase/DNase-free water. .. VN biopsy sections were then permeabilized by incubation in 70 μl of Permeabilization Mixture 1 [0.1% pepsin (#P7000-25G, Sigma-Aldrich), 0.1 M HCl (#318965-1000 ML, Sigma-Aldrich)] at 37 °C for 1 minute, and RAA biopsy sections by incubation in Permeabilization Mixture 2 [0.2% pepsin, 0.1 M HCl] at 37 °C for 15 minutes.

    Article Title: Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform
    Article Snippet: Northern blot analyses Total RNA from HAP1 cells (isolated as described above) was incubated with 75 or 150 pmol recombinant hENDOV as described above for the activity assays. .. A sample with only RNA (no enzyme) and another sample with 150 pmol BSA (Bionordika/NEB, B9000S) were included as controls.

    Article Title: THZ1 reveals roles for Cdk7 in co-transcriptional capping and pausing
    Article Snippet: CMV templates −800 to +175 ( ) or +508 ( , – ) and HeLa nuclear extract (HNE) described previously ( ; , ) were incubated for 30 min in the presence of 60 mM KCl, 5 mM MgCl2 , 20 mM HEPES pH 7.6, 1 mM DTT, and 0.5 U/μl SUPERase-In (Ambion AM2696) or 1 U/μl RNaseOUT (Invitrogen 10777-019). .. Before chase, immobilized complexes were optionally isolated with high salt wash (1.6 M KCl, 20 mM HEPES pH 7.6, 1 mM DTT, and 0.02% Tween20) or low salt wash (60 mM KCl, 20 mM HEPES pH 7.6, 1 mM DTT, 0.2 mg/ml BSA (New England BioLabs B9000), and 0.02% Tween20).

    Article Title: Assessment of DNA Topoisomerase I Unwinding Activity, Radical Scavenging Capacity, and Inhibition of Breast Cancer Cell Viability of N-alkyl-acridones and N,N′-dialkyl-9,9′-biacridylidenes
    Article Snippet: We exposed 400 ng of pUC19 plasmid DNA (NEB, N3041, Ipswich, MA, USA) to varying concentrations of drug (0.01–400 µM) for 30 min at room temperature in a final volume of 20 µL containing 80 mM HEPES (pH 7.2) or 80 mM phosphate buffer (pH 5.8), 10× CutSmart® buffer (NEB, B7204), and 100× BSA (NEB, B9000). .. Added to the mixture, was 1 unit of topoisomerase I (E. coli) (NEB, M0301) and incubated for 20 min at 37 °C to ensure relaxation of the plasmid DNA.

    Activity Assay:

    Article Title: Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform
    Article Snippet: Northern blot analyses Total RNA from HAP1 cells (isolated as described above) was incubated with 75 or 150 pmol recombinant hENDOV as described above for the activity assays. .. A sample with only RNA (no enzyme) and another sample with 150 pmol BSA (Bionordika/NEB, B9000S) were included as controls.

    Western Blot:

    Article Title: Localization and expression of GABA transporters in the suprachiasmatic nucleus
    Article Snippet: Paragraph title: Western blot procedures and data analysis ... The absorbance of bovine serum albumin (BSA) standard (B9001S or B9000S, New England BioLabs) and lysate samples was measured at 595 nm in duplicates with a Shimadzu UV-1700 PharmaSpec Spectrophotometer (Shimadzu Scientific Instruments, MD).

    RNA Binding Assay:

    Article Title: RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2
    Article Snippet: RNA was incubated for 2 min at 95 °C in 10 mM Tris-HCl pH 7.5 (at 25 °C) and was then immediately snap-cooled on ice for 2 min. Next, RNA was allowed to fold for 30 min at 37 °C in binding buffer (50 mM Tris-HCl pH 7.5 at 25 °C, 200 mM KCl, 2.5 mM MgCl2 , 0.1 mM ZnCl2 , 2 mM 2-mercaptoethanol, 0.1 mg/mL bovine serum albumin (NEB #B9000S), 0.05% Nonidet P40 (Roche #11754599001) and 0.1 mg/mL fragmented yeast tRNA (Sigma #R5636)). .. For assaying the RNA-binding affinity in the presence or absence of stimulatory or substrate peptides, binding buffer was adjusted to mimic conditions used for HMTase assays with these peptides and consisted of 50 mM Tris-HCl pH 7.5 (at 25 °C), 100 mM KCl, 2 mM 2-mercaptoethanol, 0.05% Nonidet P40 (Roche #11754599001) and 0.1 mg/mL bovine serum albumin (NEB #B9000S).

    Kinase Assay:

    Article Title: Engineering proteins for allosteric control by light or ligands
    Article Snippet: .. HEPES (Sigma, cat. no. 54457-50G-F) EGTA (Sigma, cat. no. E0396) NP-40 (abcam, cat. no ab142227) Sodium fluoride (NaF) (Sigma-Aldrich, cat. no. S7920) Sodium orthovanadate (Na3 VO4 ) (Sigma-Aldrich, cat. no. 450243) MgCl2 (Sigma-Aldrich, cat. no. M8266) MnCl2 (Sigma-Aldrich, cat. no. 244589) Brij-35 (Thermo-Fischer, cat. no. 20150) KCl (Sigma-Aldrich, cat. no. P9541) NaCl (Sigma-Aldrich, cat. no. S9888) Protein G-coupled agarose beads (Millipore, cat. no. 16-266) for kinase assay Purified kinase substrate (see ) ATP (New England Biolabs, cat. no. P0756S) Bovine serum albumin (New England Biolabs, cat. no. B9000S) 1 mM rapamycin (LC Laboratories, R-5000) stock solution in ethanol 2× Laemmli SDS-PAGE protein sample buffer (Sigma-Aldrich, cat. no. S3401) Protease inhibitor cocktail tablets (Sigma-Aldrich, cat. no. 11697498001) Anti-myc antibody (Millipore, clone 4A6, cat. no. 05-724) Anti-flag antibody (Sigma-Aldrich, cat. no. F3165-1MG) .. Bacterial expression plasmid to express mutant Rho GTPase such as pGEX-4T1-Rac1 G15A (Addgene Plasmid # 69355), pGEX-4T1-Cdc42 G15A (Addgene Plasmid # 69356), and pGEX-4T1-RhoA G17A (Addgene Plasmid # 69357) Active GTPase pull-down assay kit for Rac1 (Cytoskeleton, cat. no. BK035), Cdc42 pull-down assay kit (Cytoskeleton, cat. no. BK034), and RhoA pull-down assay kit (Cytoskeleton, cat. no. BK036) LB bacterial medium (see ) IPTG (Life Technologies, cat. no. 15529019) E. coli.

    Hybridization:

    Article Title: RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples
    Article Snippet: Next, a mix containing 1 × Ampligase buffer (20 mM Tris-HCl, pH 8.3, 25 mM KCl, 10 mM MgCl2 , 0.5 mM NAD and 0.01% Triton X-100), 100 nM of each padlock probe, 50 μM dNTPs, 0.5U/μl Ampligase (Illumina, cat. no. A3210K), 50 mM KCl and 20% formamide was added into the Secure-Seal hybridization chambers (EMS, cat.no. .. Finally, samples were incubated in RCA buffer containing 1U/μl phi29 polymerase (Thermo, cat. no. EP0091), 1 × phi29 polymerase buffer, 0.25 mM dNTPs (Thermo, cat. no. R0191), 0.2 μg/μl BSA (NEB cat. no. B9000S) and 5% glycerol in DEPC-H2 O for 1 h or otherwise specified.

    Protease Inhibitor:

    Article Title: CTD-dependent and -independent mechanisms govern co-transcriptional capping of Pol II transcripts
    Article Snippet: Bovine serum albumin (20 mg/ml, cat. no. B9000S), 2x RNA loading dye (cat. no. B0363S), and yeast inorganic pyrophosphatase (100 units/ml, cat. no. NEBM2403S) were from New England Biolabs. .. Protease inhibitor for mammalian cell extracts (cat. no. P8340) and protease inhibitor cocktail for His-Tag purifications (cat. no. P8849) were from Sigma, and 10 mM THZ1 hydrochloride in DMSO (cat. no. HY-80013A) was obtained from MedChem Express.

    Article Title: Engineering proteins for allosteric control by light or ligands
    Article Snippet: .. HEPES (Sigma, cat. no. 54457-50G-F) EGTA (Sigma, cat. no. E0396) NP-40 (abcam, cat. no ab142227) Sodium fluoride (NaF) (Sigma-Aldrich, cat. no. S7920) Sodium orthovanadate (Na3 VO4 ) (Sigma-Aldrich, cat. no. 450243) MgCl2 (Sigma-Aldrich, cat. no. M8266) MnCl2 (Sigma-Aldrich, cat. no. 244589) Brij-35 (Thermo-Fischer, cat. no. 20150) KCl (Sigma-Aldrich, cat. no. P9541) NaCl (Sigma-Aldrich, cat. no. S9888) Protein G-coupled agarose beads (Millipore, cat. no. 16-266) for kinase assay Purified kinase substrate (see ) ATP (New England Biolabs, cat. no. P0756S) Bovine serum albumin (New England Biolabs, cat. no. B9000S) 1 mM rapamycin (LC Laboratories, R-5000) stock solution in ethanol 2× Laemmli SDS-PAGE protein sample buffer (Sigma-Aldrich, cat. no. S3401) Protease inhibitor cocktail tablets (Sigma-Aldrich, cat. no. 11697498001) Anti-myc antibody (Millipore, clone 4A6, cat. no. 05-724) Anti-flag antibody (Sigma-Aldrich, cat. no. F3165-1MG) .. Bacterial expression plasmid to express mutant Rho GTPase such as pGEX-4T1-Rac1 G15A (Addgene Plasmid # 69355), pGEX-4T1-Cdc42 G15A (Addgene Plasmid # 69356), and pGEX-4T1-RhoA G17A (Addgene Plasmid # 69357) Active GTPase pull-down assay kit for Rac1 (Cytoskeleton, cat. no. BK035), Cdc42 pull-down assay kit (Cytoskeleton, cat. no. BK034), and RhoA pull-down assay kit (Cytoskeleton, cat. no. BK036) LB bacterial medium (see ) IPTG (Life Technologies, cat. no. 15529019) E. coli.

    Article Title: Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells
    Article Snippet: .. Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023) 100x L-glutamine (200 mM) (Thermo Fisher Scientific, Gibco™, catalog number: 25030081) TrypLE dissociation reagent (Thermo Fisher Scientific, Gibco™, catalog number: 12605010) Phenol red solution, 0.5% (Sigma-Aldrich, catalog number: P0290) Liquid nitrogen (LN2 ) Freestyle 293 medium (Thermo Fisher Scientific, Gibco™, catalog number: 12338026) 3 M ammonium sulfate in 0.1 M sodium phosphate buffer pH 7.4 Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G5516) 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: 54457) Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014) Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 71687) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 320331) Triton X-100 (Sigma-Aldrich, catalog number: T8787) Protease inhibitor cocktail, EDTA-free (Roche Diagnostics, catalog number: 11873580001) CHAPS (Sigma-Aldrich, catalog number: C5070) Anti-FLAG M2 antibody (Sigma-Aldrich, catalog number: F3165 or F1804) Dynabeads M-270 epoxy (Thermo Fisher Scientific, Invitrogen™, catalog number: 14302D) 0.1 M sodium phosphate buffer pH 7.4 Tris base (Sigma-Aldrich, catalog number: 93362) 3xFLAG peptide (Sigma-Aldrich, catalog number: F4799) reconstituted at 5 mg/ml in TBS (50 mM Tris-HCl pH 7.4, 150 mM NaCl). .. Aliquots should be stored at −20 °C DTSSP (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 21578) 4x LDS (lithium dodecyl sulfate) sample loading buffer (Thermo Fisher Scientific, Novex™, catalog number: NP0007) 10x sample reducing agent (Thermo Fisher Scientific, Novex™, catalog number: NP0004); or 500 mM dithiothreitol (DTT) 4–12% Bis-Tris 26-well midi gels (Thermo Fisher Scientific, Invitrogen™, catalog number: WG1403BOX) 20x MOPS (3-morpholinopropane-1-sulfonic acid) gel running buffer (Thermo Fisher Scientific, Novex™, catalog number: NP0001) SilverQuest Silver Staining Kit (Thermo Fisher Scientific, Novex™, catalog number: LC6070) Sypro ruby protein gel stain (Sigma-Aldrich, catalog number: S4942) Anti-EXOSC10 antibody (Abcam, catalog number: ab95028) Anti-SKIV2L2 antibody (Abcam, catalog number: ab70552) Anti-EXOSC3 antibody (Proteintech, catalog number: 15062-1-AP) ExoI extraction solution (see Recipes) ExoII extraction solution (see Recipes) Gradient solution – ‘light’ (see Recipes) Gradient solution – ‘heavy’ (see Recipes)

    Transferring:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: Eppendorf tubes (1.5 ml) Pipette tips (10 μl, 100 μl, 1,000 μl) Deoxyguanosine triphosphate (dGTP) (New England Biolabs, catalog number: N0447S) 8-oxo-2′-deoxyguanosine-5′-Triphosphate (8-oxo-dGTP) (Jena Bioscience, catalog number: NU-1117L) DNA substrate: The DNA substrate includes a fluorescent tag at both the 5′- and 3′-ends of one of the gap-containing strand in the double-stranded DNA with a single nucleotide gap opposite template base Cytosine ( ) Note: The sequence information for the DNA substrate is presented in the Notes section of the protocol. .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes)

    Northern Blot:

    Article Title: Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform
    Article Snippet: Paragraph title: Northern blot analyses ... A sample with only RNA (no enzyme) and another sample with 150 pmol BSA (Bionordika/NEB, B9000S) were included as controls.

    other:

    Article Title: Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry
    Article Snippet: RNAP holoenzyme was reconstituted by mixing Alexa 647-labeled RNAP core (4 μL of 0.5 μM) and sigma-70 (either 4 μL of 4 μM WT unlabeled or 2 μL of 1.9 μM C132 Cy3B-labeled), bringing the volume to 24 μL with 1 × transcription buffer (50mM Tris-acetate pH 8.0, 200mM potassium acetate, 8mM magnesium acetate, 27.6mM ammonium acetate, 10 μg/mL BSA NEB B9000S, 10mM DTT) and incubating for 15 min to 2 hr at 37°C.

    Protein Concentration:

    Article Title: RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2
    Article Snippet: RNA was incubated for 2 min at 95 °C in 10 mM Tris-HCl pH 7.5 (at 25 °C) and was then immediately snap-cooled on ice for 2 min. Next, RNA was allowed to fold for 30 min at 37 °C in binding buffer (50 mM Tris-HCl pH 7.5 at 25 °C, 200 mM KCl, 2.5 mM MgCl2 , 0.1 mM ZnCl2 , 2 mM 2-mercaptoethanol, 0.1 mg/mL bovine serum albumin (NEB #B9000S), 0.05% Nonidet P40 (Roche #11754599001) and 0.1 mg/mL fragmented yeast tRNA (Sigma #R5636)). .. Serial dilutions of the protein were made separately, in the same buffer, and combined with the RNA solution for a final reaction volume of 40 μL containing 5 nM fluorescently-labelled RNA and the desired final protein concentration.

    Sequencing:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: Eppendorf tubes (1.5 ml) Pipette tips (10 μl, 100 μl, 1,000 μl) Deoxyguanosine triphosphate (dGTP) (New England Biolabs, catalog number: N0447S) 8-oxo-2′-deoxyguanosine-5′-Triphosphate (8-oxo-dGTP) (Jena Bioscience, catalog number: NU-1117L) DNA substrate: The DNA substrate includes a fluorescent tag at both the 5′- and 3′-ends of one of the gap-containing strand in the double-stranded DNA with a single nucleotide gap opposite template base Cytosine ( ) Note: The sequence information for the DNA substrate is presented in the Notes section of the protocol. .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes)

    Sonication:

    Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation
    Article Snippet: .. Diluted chromatin samples were pre-cleared for 1 h with 55 μL of 50 % suspension of protein A sepharose (Protein A Sepharose CL-4B, GE Healthcare N: 17-0780-01) beads that were blocked with 0.2 μg/μL sonicated herring sperm DNA (Thermo Fisher Scientific, N: 15634-017) and 0.5 μg/μL BSA (B9000 S, NEB). ..

    Binding Assay:

    Article Title: RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2
    Article Snippet: .. RNA was incubated for 2 min at 95 °C in 10 mM Tris-HCl pH 7.5 (at 25 °C) and was then immediately snap-cooled on ice for 2 min. Next, RNA was allowed to fold for 30 min at 37 °C in binding buffer (50 mM Tris-HCl pH 7.5 at 25 °C, 200 mM KCl, 2.5 mM MgCl2 , 0.1 mM ZnCl2 , 2 mM 2-mercaptoethanol, 0.1 mg/mL bovine serum albumin (NEB #B9000S), 0.05% Nonidet P40 (Roche #11754599001) and 0.1 mg/mL fragmented yeast tRNA (Sigma #R5636)). .. For assaying the RNA-binding affinity in the presence or absence of stimulatory or substrate peptides, binding buffer was adjusted to mimic conditions used for HMTase assays with these peptides and consisted of 50 mM Tris-HCl pH 7.5 (at 25 °C), 100 mM KCl, 2 mM 2-mercaptoethanol, 0.05% Nonidet P40 (Roche #11754599001) and 0.1 mg/mL bovine serum albumin (NEB #B9000S).

    Fluorescence:

    Article Title: RNA exploits an exposed regulatory site to inhibit the enzymatic activity of PRC2
    Article Snippet: Paragraph title: Fluorescence anisotropy assay ... RNA was incubated for 2 min at 95 °C in 10 mM Tris-HCl pH 7.5 (at 25 °C) and was then immediately snap-cooled on ice for 2 min. Next, RNA was allowed to fold for 30 min at 37 °C in binding buffer (50 mM Tris-HCl pH 7.5 at 25 °C, 200 mM KCl, 2.5 mM MgCl2 , 0.1 mM ZnCl2 , 2 mM 2-mercaptoethanol, 0.1 mg/mL bovine serum albumin (NEB #B9000S), 0.05% Nonidet P40 (Roche #11754599001) and 0.1 mg/mL fragmented yeast tRNA (Sigma #R5636)).

    Isolation:

    Article Title: Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform
    Article Snippet: Northern blot analyses Total RNA from HAP1 cells (isolated as described above) was incubated with 75 or 150 pmol recombinant hENDOV as described above for the activity assays. .. A sample with only RNA (no enzyme) and another sample with 150 pmol BSA (Bionordika/NEB, B9000S) were included as controls.

    Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation
    Article Snippet: Chromatin immunoprecipitation (ChIP) Chromatin was isolated from TIS7 WT and KO formaldehyde-treated MSCs using the EpiSeeker Chromatin Extraction Kit (ab117152, Abcam) according to the manufacturer’s protocol. .. Diluted chromatin samples were pre-cleared for 1 h with 55 μL of 50 % suspension of protein A sepharose (Protein A Sepharose CL-4B, GE Healthcare N: 17-0780-01) beads that were blocked with 0.2 μg/μL sonicated herring sperm DNA (Thermo Fisher Scientific, N: 15634-017) and 0.5 μg/μL BSA (B9000 S, NEB).

    Article Title: THZ1 reveals roles for Cdk7 in co-transcriptional capping and pausing
    Article Snippet: .. Before chase, immobilized complexes were optionally isolated with high salt wash (1.6 M KCl, 20 mM HEPES pH 7.6, 1 mM DTT, and 0.02% Tween20) or low salt wash (60 mM KCl, 20 mM HEPES pH 7.6, 1 mM DTT, 0.2 mg/ml BSA (New England BioLabs B9000), and 0.02% Tween20). .. Labeled transcripts were phenol extracted, ethanol precipitated and separated on denaturing RNA gels.

    Article Title: A retained intron in the 3′‐ UTR of Calm3 mRNA mediates its Staufen2‐ and activity‐dependent localization to neuronal dendrites
    Article Snippet: For cDNA synthesis using total RNA isolated from rat cortical neurons, 2 μg total RNA for each sample was treated with 1 unit of DNase I (Thermo Fisher Scientific) at 37°C for 30 min. DNase‐treated RNA was split in two: 1 μg was used for cDNA synthesis and the rest 1 μg for minus reverse transcriptase (−RT) reactions. .. For detecting mRNA levels by qPCR, a homemade SYBR Green Mix [containing the following components at a final concentration of 1 M betaine (B0300; Sigma), 1× standard Taq buffer (NEB), 16 μM dNTPs (N0447S; NEB), BSA 20 μg/ml (B9000S; NEB), 0.6 U per reaction Hot‐Start Taq DNA polymerase (M0495S; NEB), and 1 μl/ml of 1:100 SYBR Green (20010; Lumiprobe)] (in ddH2 O) was used.

    Size-exclusion Chromatography:

    Article Title: THZ1 reveals roles for Cdk7 in co-transcriptional capping and pausing
    Article Snippet: Initiation was accomplished with a 30 or 45 sec pulse containing 60 mM KCl, 5 mM MgCl2 , 20 mM HEPES pH 7.6, 0.21 μM α-32 P-CTP, and 500 μM ATP/UTP/GTP (limiting CTP) or 500 μM ATP/GTP 0.1 μM UTP (limiting UTP/CTP). .. Before chase, immobilized complexes were optionally isolated with high salt wash (1.6 M KCl, 20 mM HEPES pH 7.6, 1 mM DTT, and 0.02% Tween20) or low salt wash (60 mM KCl, 20 mM HEPES pH 7.6, 1 mM DTT, 0.2 mg/ml BSA (New England BioLabs B9000), and 0.02% Tween20).

    Labeling:

    Article Title: THZ1 reveals roles for Cdk7 in co-transcriptional capping and pausing
    Article Snippet: Before chase, immobilized complexes were optionally isolated with high salt wash (1.6 M KCl, 20 mM HEPES pH 7.6, 1 mM DTT, and 0.02% Tween20) or low salt wash (60 mM KCl, 20 mM HEPES pH 7.6, 1 mM DTT, 0.2 mg/ml BSA (New England BioLabs B9000), and 0.02% Tween20). .. Labeled transcripts were phenol extracted, ethanol precipitated and separated on denaturing RNA gels.

    Purification:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes) .. Pipettes (P2, P10, P20, P100, P200) Table-top heat block (Digital Dry Block Heater, VWR) Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2) Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500)

    Article Title: Engineering proteins for allosteric control by light or ligands
    Article Snippet: .. HEPES (Sigma, cat. no. 54457-50G-F) EGTA (Sigma, cat. no. E0396) NP-40 (abcam, cat. no ab142227) Sodium fluoride (NaF) (Sigma-Aldrich, cat. no. S7920) Sodium orthovanadate (Na3 VO4 ) (Sigma-Aldrich, cat. no. 450243) MgCl2 (Sigma-Aldrich, cat. no. M8266) MnCl2 (Sigma-Aldrich, cat. no. 244589) Brij-35 (Thermo-Fischer, cat. no. 20150) KCl (Sigma-Aldrich, cat. no. P9541) NaCl (Sigma-Aldrich, cat. no. S9888) Protein G-coupled agarose beads (Millipore, cat. no. 16-266) for kinase assay Purified kinase substrate (see ) ATP (New England Biolabs, cat. no. P0756S) Bovine serum albumin (New England Biolabs, cat. no. B9000S) 1 mM rapamycin (LC Laboratories, R-5000) stock solution in ethanol 2× Laemmli SDS-PAGE protein sample buffer (Sigma-Aldrich, cat. no. S3401) Protease inhibitor cocktail tablets (Sigma-Aldrich, cat. no. 11697498001) Anti-myc antibody (Millipore, clone 4A6, cat. no. 05-724) Anti-flag antibody (Sigma-Aldrich, cat. no. F3165-1MG) .. Bacterial expression plasmid to express mutant Rho GTPase such as pGEX-4T1-Rac1 G15A (Addgene Plasmid # 69355), pGEX-4T1-Cdc42 G15A (Addgene Plasmid # 69356), and pGEX-4T1-RhoA G17A (Addgene Plasmid # 69357) Active GTPase pull-down assay kit for Rac1 (Cytoskeleton, cat. no. BK035), Cdc42 pull-down assay kit (Cytoskeleton, cat. no. BK034), and RhoA pull-down assay kit (Cytoskeleton, cat. no. BK036) LB bacterial medium (see ) IPTG (Life Technologies, cat. no. 15529019) E. coli.

    Polymerase Chain Reaction:

    Article Title: A retained intron in the 3′‐ UTR of Calm3 mRNA mediates its Staufen2‐ and activity‐dependent localization to neuronal dendrites
    Article Snippet: To detect Calm1 , Calm2 , and Calm3 mRNAs, quantitative reverse transcriptase PCR (qRT–PCR) was performed using the SYBR Green Master Mix (Bio‐Rad) according to the manufacturer's instructions. .. For detecting mRNA levels by qPCR, a homemade SYBR Green Mix [containing the following components at a final concentration of 1 M betaine (B0300; Sigma), 1× standard Taq buffer (NEB), 16 μM dNTPs (N0447S; NEB), BSA 20 μg/ml (B9000S; NEB), 0.6 U per reaction Hot‐Start Taq DNA polymerase (M0495S; NEB), and 1 μl/ml of 1:100 SYBR Green (20010; Lumiprobe)] (in ddH2 O) was used.

    Polyacrylamide Gel Electrophoresis:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes) .. Pipettes (P2, P10, P20, P100, P200) Table-top heat block (Digital Dry Block Heater, VWR) Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2) Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500)

    Article Title: Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform
    Article Snippet: A sample with only RNA (no enzyme) and another sample with 150 pmol BSA (Bionordika/NEB, B9000S) were included as controls. .. Equal volume of formamide loading dye was added and the samples were heated at 50°C for 5 min before separation by 15% denaturating PAGE (7 M urea and 1x taurine) at 200 V for 50 min in 1x taurine.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples
    Article Snippet: Next, a mix containing 1 × Ampligase buffer (20 mM Tris-HCl, pH 8.3, 25 mM KCl, 10 mM MgCl2 , 0.5 mM NAD and 0.01% Triton X-100), 100 nM of each padlock probe, 50 μM dNTPs, 0.5U/μl Ampligase (Illumina, cat. no. A3210K), 50 mM KCl and 20% formamide was added into the Secure-Seal hybridization chambers (EMS, cat.no. .. Finally, samples were incubated in RCA buffer containing 1U/μl phi29 polymerase (Thermo, cat. no. EP0091), 1 × phi29 polymerase buffer, 0.25 mM dNTPs (Thermo, cat. no. R0191), 0.2 μg/μl BSA (NEB cat. no. B9000S) and 5% glycerol in DEPC-H2 O for 1 h or otherwise specified.

    Silver Staining:

    Article Title: Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells
    Article Snippet: Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023) 100x L-glutamine (200 mM) (Thermo Fisher Scientific, Gibco™, catalog number: 25030081) TrypLE dissociation reagent (Thermo Fisher Scientific, Gibco™, catalog number: 12605010) Phenol red solution, 0.5% (Sigma-Aldrich, catalog number: P0290) Liquid nitrogen (LN2 ) Freestyle 293 medium (Thermo Fisher Scientific, Gibco™, catalog number: 12338026) 3 M ammonium sulfate in 0.1 M sodium phosphate buffer pH 7.4 Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G5516) 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: 54457) Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014) Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 71687) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 320331) Triton X-100 (Sigma-Aldrich, catalog number: T8787) Protease inhibitor cocktail, EDTA-free (Roche Diagnostics, catalog number: 11873580001) CHAPS (Sigma-Aldrich, catalog number: C5070) Anti-FLAG M2 antibody (Sigma-Aldrich, catalog number: F3165 or F1804) Dynabeads M-270 epoxy (Thermo Fisher Scientific, Invitrogen™, catalog number: 14302D) 0.1 M sodium phosphate buffer pH 7.4 Tris base (Sigma-Aldrich, catalog number: 93362) 3xFLAG peptide (Sigma-Aldrich, catalog number: F4799) reconstituted at 5 mg/ml in TBS (50 mM Tris-HCl pH 7.4, 150 mM NaCl). .. Aliquots should be stored at −20 °C DTSSP (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 21578) 4x LDS (lithium dodecyl sulfate) sample loading buffer (Thermo Fisher Scientific, Novex™, catalog number: NP0007) 10x sample reducing agent (Thermo Fisher Scientific, Novex™, catalog number: NP0004); or 500 mM dithiothreitol (DTT) 4–12% Bis-Tris 26-well midi gels (Thermo Fisher Scientific, Invitrogen™, catalog number: WG1403BOX) 20x MOPS (3-morpholinopropane-1-sulfonic acid) gel running buffer (Thermo Fisher Scientific, Novex™, catalog number: NP0001) SilverQuest Silver Staining Kit (Thermo Fisher Scientific, Novex™, catalog number: LC6070) Sypro ruby protein gel stain (Sigma-Aldrich, catalog number: S4942) Anti-EXOSC10 antibody (Abcam, catalog number: ab95028) Anti-SKIV2L2 antibody (Abcam, catalog number: ab70552) Anti-EXOSC3 antibody (Proteintech, catalog number: 15062-1-AP) ExoI extraction solution (see Recipes) ExoII extraction solution (see Recipes) Gradient solution – ‘light’ (see Recipes) Gradient solution – ‘heavy’ (see Recipes)

    Chromatin Immunoprecipitation:

    Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation
    Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) ... Diluted chromatin samples were pre-cleared for 1 h with 55 μL of 50 % suspension of protein A sepharose (Protein A Sepharose CL-4B, GE Healthcare N: 17-0780-01) beads that were blocked with 0.2 μg/μL sonicated herring sperm DNA (Thermo Fisher Scientific, N: 15634-017) and 0.5 μg/μL BSA (B9000 S, NEB).

    SDS Page:

    Article Title: Engineering proteins for allosteric control by light or ligands
    Article Snippet: .. HEPES (Sigma, cat. no. 54457-50G-F) EGTA (Sigma, cat. no. E0396) NP-40 (abcam, cat. no ab142227) Sodium fluoride (NaF) (Sigma-Aldrich, cat. no. S7920) Sodium orthovanadate (Na3 VO4 ) (Sigma-Aldrich, cat. no. 450243) MgCl2 (Sigma-Aldrich, cat. no. M8266) MnCl2 (Sigma-Aldrich, cat. no. 244589) Brij-35 (Thermo-Fischer, cat. no. 20150) KCl (Sigma-Aldrich, cat. no. P9541) NaCl (Sigma-Aldrich, cat. no. S9888) Protein G-coupled agarose beads (Millipore, cat. no. 16-266) for kinase assay Purified kinase substrate (see ) ATP (New England Biolabs, cat. no. P0756S) Bovine serum albumin (New England Biolabs, cat. no. B9000S) 1 mM rapamycin (LC Laboratories, R-5000) stock solution in ethanol 2× Laemmli SDS-PAGE protein sample buffer (Sigma-Aldrich, cat. no. S3401) Protease inhibitor cocktail tablets (Sigma-Aldrich, cat. no. 11697498001) Anti-myc antibody (Millipore, clone 4A6, cat. no. 05-724) Anti-flag antibody (Sigma-Aldrich, cat. no. F3165-1MG) .. Bacterial expression plasmid to express mutant Rho GTPase such as pGEX-4T1-Rac1 G15A (Addgene Plasmid # 69355), pGEX-4T1-Cdc42 G15A (Addgene Plasmid # 69356), and pGEX-4T1-RhoA G17A (Addgene Plasmid # 69357) Active GTPase pull-down assay kit for Rac1 (Cytoskeleton, cat. no. BK035), Cdc42 pull-down assay kit (Cytoskeleton, cat. no. BK034), and RhoA pull-down assay kit (Cytoskeleton, cat. no. BK036) LB bacterial medium (see ) IPTG (Life Technologies, cat. no. 15529019) E. coli.

    Article Title: Localization and expression of GABA transporters in the suprachiasmatic nucleus
    Article Snippet: The absorbance of bovine serum albumin (BSA) standard (B9001S or B9000S, New England BioLabs) and lysate samples was measured at 595 nm in duplicates with a Shimadzu UV-1700 PharmaSpec Spectrophotometer (Shimadzu Scientific Instruments, MD). .. During the SDS-PAGE routine, the samples of SCN tissue, that represented different ZTs, were run simultaneously on the same gel.

    Plasmid Preparation:

    Article Title: Assessment of DNA Topoisomerase I Unwinding Activity, Radical Scavenging Capacity, and Inhibition of Breast Cancer Cell Viability of N-alkyl-acridones and N,N′-dialkyl-9,9′-biacridylidenes
    Article Snippet: .. We exposed 400 ng of pUC19 plasmid DNA (NEB, N3041, Ipswich, MA, USA) to varying concentrations of drug (0.01–400 µM) for 30 min at room temperature in a final volume of 20 µL containing 80 mM HEPES (pH 7.2) or 80 mM phosphate buffer (pH 5.8), 10× CutSmart® buffer (NEB, B7204), and 100× BSA (NEB, B9000). .. Added to the mixture, was 1 unit of topoisomerase I (E. coli) (NEB, M0301) and incubated for 20 min at 37 °C to ensure relaxation of the plasmid DNA.

    SYBR Green Assay:

    Article Title: A retained intron in the 3′‐ UTR of Calm3 mRNA mediates its Staufen2‐ and activity‐dependent localization to neuronal dendrites
    Article Snippet: .. For detecting mRNA levels by qPCR, a homemade SYBR Green Mix [containing the following components at a final concentration of 1 M betaine (B0300; Sigma), 1× standard Taq buffer (NEB), 16 μM dNTPs (N0447S; NEB), BSA 20 μg/ml (B9000S; NEB), 0.6 U per reaction Hot‐Start Taq DNA polymerase (M0495S; NEB), and 1 μl/ml of 1:100 SYBR Green (20010; Lumiprobe)] (in ddH2 O) was used. .. Forward and reverse primer pair mix was used 2 μl per reaction from the following stock concentrations: Renilla and firefly luciferase at 3 μM, GFP 5 μM, and Calm3 ORF Fwd/Rev 2.5 μM; Calm3 ORF Fwd/Calm3 intron Rev 4 μM, Stau2 5 μM, pp1a 3 μM, GFP Fwd/Calm3 intron Rev 4 μM, and Renilla luciferase Fwd/Calm3 intron Rev 4 μM.

    Recombinant:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes) .. Pipettes (P2, P10, P20, P100, P200) Table-top heat block (Digital Dry Block Heater, VWR) Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2) Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500)

    Article Title: Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform
    Article Snippet: Northern blot analyses Total RNA from HAP1 cells (isolated as described above) was incubated with 75 or 150 pmol recombinant hENDOV as described above for the activity assays. .. A sample with only RNA (no enzyme) and another sample with 150 pmol BSA (Bionordika/NEB, B9000S) were included as controls.

    In Vitro:

    Article Title: THZ1 reveals roles for Cdk7 in co-transcriptional capping and pausing
    Article Snippet: Paragraph title: In vitro transcription ... Before chase, immobilized complexes were optionally isolated with high salt wash (1.6 M KCl, 20 mM HEPES pH 7.6, 1 mM DTT, and 0.02% Tween20) or low salt wash (60 mM KCl, 20 mM HEPES pH 7.6, 1 mM DTT, 0.2 mg/ml BSA (New England BioLabs B9000), and 0.02% Tween20).

    Spectrophotometry:

    Article Title: Localization and expression of GABA transporters in the suprachiasmatic nucleus
    Article Snippet: .. The absorbance of bovine serum albumin (BSA) standard (B9001S or B9000S, New England BioLabs) and lysate samples was measured at 595 nm in duplicates with a Shimadzu UV-1700 PharmaSpec Spectrophotometer (Shimadzu Scientific Instruments, MD). ..

    Concentration Assay:

    Article Title: Assessment of DNA Topoisomerase I Unwinding Activity, Radical Scavenging Capacity, and Inhibition of Breast Cancer Cell Viability of N-alkyl-acridones and N,N′-dialkyl-9,9′-biacridylidenes
    Article Snippet: We exposed 400 ng of pUC19 plasmid DNA (NEB, N3041, Ipswich, MA, USA) to varying concentrations of drug (0.01–400 µM) for 30 min at room temperature in a final volume of 20 µL containing 80 mM HEPES (pH 7.2) or 80 mM phosphate buffer (pH 5.8), 10× CutSmart® buffer (NEB, B7204), and 100× BSA (NEB, B9000). .. The reaction was stopped through the addition of SDS and protein kinase, to a final concentration of 0.25% and 250 µg/mL, respectively.

    Article Title: A retained intron in the 3′‐ UTR of Calm3 mRNA mediates its Staufen2‐ and activity‐dependent localization to neuronal dendrites
    Article Snippet: .. For detecting mRNA levels by qPCR, a homemade SYBR Green Mix [containing the following components at a final concentration of 1 M betaine (B0300; Sigma), 1× standard Taq buffer (NEB), 16 μM dNTPs (N0447S; NEB), BSA 20 μg/ml (B9000S; NEB), 0.6 U per reaction Hot‐Start Taq DNA polymerase (M0495S; NEB), and 1 μl/ml of 1:100 SYBR Green (20010; Lumiprobe)] (in ddH2 O) was used. .. Forward and reverse primer pair mix was used 2 μl per reaction from the following stock concentrations: Renilla and firefly luciferase at 3 μM, GFP 5 μM, and Calm3 ORF Fwd/Rev 2.5 μM; Calm3 ORF Fwd/Calm3 intron Rev 4 μM, Stau2 5 μM, pp1a 3 μM, GFP Fwd/Calm3 intron Rev 4 μM, and Renilla luciferase Fwd/Calm3 intron Rev 4 μM.

    Marker:

    Article Title: In vitro Assay to Measure DNA Polymerase β Nucleotide Insertion Coupled with the DNA Ligation Reaction during Base Excision Repair
    Article Snippet: .. Tris-HCl (pH 7.5) Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333) Magnesium chloride (MgCl2 ) (Sigma-Aldrich, catalog number: M8266) Adenosine 5′-Triphosphate (ATP) (New England Biolabs, catalog number: P0756S) Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632) Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G9012) HEPES (pH 7.5) Sodium chloride (NaCl) EDTA (Sigma-Aldrich, catalog number: 93283) Purified enzymes: Recombinant human DNA polymerase β and DNA ligase I (see Recipes and ) Formamide (Sigma-Aldrich, catalog number: F9037) Bromophenol blue (Sigma-Aldrich, catalog number: B0126) Xylene cyanol (Sigma-Aldrich, catalog number: X4126) Trizma-base (Sigma-Aldrich, catalog number: T4661) Boric acid (Promega, catalog number: H5003) Urea (National Diagnostic, catalog number: EC-605) Ammonium persulfate (APS) (Sigma-Aldrich, catalog number: A3678-25G) Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML) Sterile water AccuGel (40%) 19:1 Acrylamide to Bisacrylamide Stabilized Solution (National Diagnostic, catalog number: EC-850) Full-range rainbow protein ladder (Amersham™ ECL™ Rainbow™ Marker) (GE Healthcare, catalog number: RPN800E) 1× reaction buffer (see Recipes) 1× protein storage and dilution buffer (see Recipes) Enzyme mixture (see Recipes) Gel-loading dye (see Recipes) 10× TBE solution (see Recipes) Denaturing PAGE solution (15%) (see Recipes) EDTA buffer (pH 8.0) (see Recipes) .. Pipettes (P2, P10, P20, P100, P200) Table-top heat block (Digital Dry Block Heater, VWR) Polyacrylamide gel electrophoresis (PAGE) apparatus (Biometra, model: Model S2) Typhoon Phosphor Imager (GE Healthcare, model: Typhoon FLA 9500)

    Staining:

    Article Title: Complex alternative splicing of human Endonuclease V mRNA, but evidence for only a single protein isoform
    Article Snippet: A sample with only RNA (no enzyme) and another sample with 150 pmol BSA (Bionordika/NEB, B9000S) were included as controls. .. One of the gels was stained with ethidium bromide to check for RNA integrity.

    Article Title: Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells
    Article Snippet: Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023) 100x L-glutamine (200 mM) (Thermo Fisher Scientific, Gibco™, catalog number: 25030081) TrypLE dissociation reagent (Thermo Fisher Scientific, Gibco™, catalog number: 12605010) Phenol red solution, 0.5% (Sigma-Aldrich, catalog number: P0290) Liquid nitrogen (LN2 ) Freestyle 293 medium (Thermo Fisher Scientific, Gibco™, catalog number: 12338026) 3 M ammonium sulfate in 0.1 M sodium phosphate buffer pH 7.4 Bovine serum albumin (BSA) (New England Biolabs, catalog number: B9000S) Glycerol (Sigma-Aldrich, catalog number: G5516) 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: 54457) Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014) Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 71687) Hydrochloric acid (HCl) (Sigma-Aldrich, catalog number: 320331) Triton X-100 (Sigma-Aldrich, catalog number: T8787) Protease inhibitor cocktail, EDTA-free (Roche Diagnostics, catalog number: 11873580001) CHAPS (Sigma-Aldrich, catalog number: C5070) Anti-FLAG M2 antibody (Sigma-Aldrich, catalog number: F3165 or F1804) Dynabeads M-270 epoxy (Thermo Fisher Scientific, Invitrogen™, catalog number: 14302D) 0.1 M sodium phosphate buffer pH 7.4 Tris base (Sigma-Aldrich, catalog number: 93362) 3xFLAG peptide (Sigma-Aldrich, catalog number: F4799) reconstituted at 5 mg/ml in TBS (50 mM Tris-HCl pH 7.4, 150 mM NaCl). .. Aliquots should be stored at −20 °C DTSSP (Thermo Fisher Scientific, Thermo Scientific™, catalog number: 21578) 4x LDS (lithium dodecyl sulfate) sample loading buffer (Thermo Fisher Scientific, Novex™, catalog number: NP0007) 10x sample reducing agent (Thermo Fisher Scientific, Novex™, catalog number: NP0004); or 500 mM dithiothreitol (DTT) 4–12% Bis-Tris 26-well midi gels (Thermo Fisher Scientific, Invitrogen™, catalog number: WG1403BOX) 20x MOPS (3-morpholinopropane-1-sulfonic acid) gel running buffer (Thermo Fisher Scientific, Novex™, catalog number: NP0001) SilverQuest Silver Staining Kit (Thermo Fisher Scientific, Novex™, catalog number: LC6070) Sypro ruby protein gel stain (Sigma-Aldrich, catalog number: S4942) Anti-EXOSC10 antibody (Abcam, catalog number: ab95028) Anti-SKIV2L2 antibody (Abcam, catalog number: ab70552) Anti-EXOSC3 antibody (Proteintech, catalog number: 15062-1-AP) ExoI extraction solution (see Recipes) ExoII extraction solution (see Recipes) Gradient solution – ‘light’ (see Recipes) Gradient solution – ‘heavy’ (see Recipes)

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    New England Biolabs low salt wash
    Low Salt Wash, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/low salt wash/product/New England Biolabs
    Average 90 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    low salt wash - by Bioz Stars, 2020-01
    90/100 stars
      Buy from Supplier

    90
    New England Biolabs n3041l bsa i hf
    N3041l Bsa I Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/n3041l bsa i hf/product/New England Biolabs
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    n3041l bsa i hf - by Bioz Stars, 2020-01
    90/100 stars
      Buy from Supplier

    Image Search Results