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Simultaneous optimization of <t>EDTA,</t> <t>NaCl,</t> and SDS concentrations enhances protein yield during solubilization of TRIzol-precipitated protein
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1) Product Images from "Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue"

Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue

Journal: Journal of neuroscience methods

doi: 10.1016/j.jneumeth.2017.02.002

Simultaneous optimization of EDTA, NaCl, and SDS concentrations enhances protein yield during solubilization of TRIzol-precipitated protein
Figure Legend Snippet: Simultaneous optimization of EDTA, NaCl, and SDS concentrations enhances protein yield during solubilization of TRIzol-precipitated protein

Techniques Used:

3.1 EDTA, NaCl, and SDS concentrations modulate protein yield during solubilization of TRIzol-precipitated protein
Figure Legend Snippet: 3.1 EDTA, NaCl, and SDS concentrations modulate protein yield during solubilization of TRIzol-precipitated protein

Techniques Used:

EDTA, NaCl, and SDS concentrations significantly alter protein yield during solubilization of TRIzol-precipitated protein
Figure Legend Snippet: EDTA, NaCl, and SDS concentrations significantly alter protein yield during solubilization of TRIzol-precipitated protein

Techniques Used:

2) Product Images from "Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy"

Article Title: Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy

Journal: Current Biology

doi: 10.1016/j.cub.2017.06.021

Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .
Figure Legend Snippet: Mib1 E3 Ligase Interacts with and Destabilizes GABARAP and Promotes GABARAP Ubiquitination at Lys13 and Lys23 (A) HEK293A cells expressing FLAG-Mib1 or control vector for 48 hr were analyzed by immunoblot. (B) Quantification of (A). Statistical analysis using unpaired Student’s t test; mean ± SEM; n = 3; ∗ p ≤ 0.001. (C) Anti-GABARAP immunoprecipitate from HEK293A cells analyzed by immunoblotting. Ab, anti-GABARAP antibody. Lys, HEK293A lysate. (D) HEK293A cells expressing FLAG-tagged constructs were incubated with recombinant GST or GST-GABARAP beads and immunoblotted. (E) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were stringently washed in denaturing buffer. CS, C985S; GAB, GABARAP; Ponc, Ponceau S. Short and long exposures are shown. ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-GABARAP, respectively. (F) Immunoprecipitation of U2OS cells expressing the indicated constructs lysed in boiling SDS buffer and immunoblot. Free ubiquitin and ∗ , ∗∗ , ∗∗∗ , mono-, di-, and tri-ubiquitinated GFP-LC3B/GABARAP are indicated, respectively. (G) GFP-TRAP of HEK293A cells expressing the indicated constructs and immunoblot. Immunoprecipitates were washed as in (E). (H) See (G). Low and high exposures are shown. (I) Immunoprecipitation of HEK293A cells expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% w/v Triton X-100, 5 mM EDTA) + N-ethylmaleimide. Diubiquitinated GABARAP is indicated with ∗∗ . Immunoglobulin light chain is indicated with an arrow. (J) Immunoprecipitation of HEK293A cells treated with RF or GABARAP siRNA for 72 hr and expressing the indicated constructs and immunoblot. Cells were treated with MG132 for 5 hr prior to lysis in TNTE buffer + N-ethylmaleimide. Di- and tri-ubiquitinated GABARAP is indicated with ∗∗ and ∗∗∗ , respectively. Immunoglobulin light chain is indicated with an arrow. (K) Two GABARAP ubiquitination sites, lysine 13 (K13) and lysine 23 (K23), were identified by mass spectrometry on three different peptides. (Top) Comparison of peak areas for the FVYKEEHPFEK(diGly)R peptide containing K13 ubiquitination site (n = 3 measurements) is shown. (Middle and bottom) The K23 ubiquitination site was detected as two different peptides as a result of missed cleavage. Quantification of peptides K(diGly)KYPDRVPVIVEK (middle) and K(diGly)KYPDR (bottom) showed significantly lower abundance in C985S mutant compared to the WT. (L) Conservation of GABARAP K13 and K23 ( ∗ ) between ATG8 orthologs. See also Figure S5 .

Techniques Used: Expressing, Plasmid Preparation, Construct, Incubation, Recombinant, Immunoprecipitation, Lysis, Mass Spectrometry, Mutagenesis

3) Product Images from "Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients"

Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

Journal: Journal of Virology

doi: 10.1128/JVI.00798-18

CsCl density gradient separation and analysis of viral particles from HepAD38 cell culture supernatant. (A) Native agarose gel analysis of viral particles. Culture supernatant of HepAD38 cells was concentrated (via ultrafiltration) and fractionated by CsCl density gradient centrifugation (3 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 1.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral particles in each fraction were resolved by native agarose gel electrophoresis, followed by detection of viral antigens with anti-HBsAg and anti-HBcAg antibodies and viral DNA by hybridization with minus-strand-specific riboprobe. (B to F) Southern and Northern blot detection of viral nucleic acids. Viral DNAs were separated by electrophoresis through Tris-acetate-EDTA (TAE) or alkaline (ALK) agarose gel for Southern blotting with minus- or plus-strand-specific riboprobes. Viral RNA was obtained by treatment with total nucleic acids with DNase I and separated by formaldehyde-MOPS agarose gel, followed by Northern blotting. (G) Quantification of viral DNA and RNA in naked capsids or virions. Fractions containing naked capsids (fractions 3 to 7) or virions (fractions 10 to 21) were pooled, and viral DNA and RNA were quantified by PCR. (H) DNA and RNA ratios in naked capsids and virions calculated based on quantitative results. Asterisks indicate unknown high-density viral particles detected by anti-HBcAg or anti-HBsAg antibodies but devoid of any HBV-specific nucleic acids. M, markers ( E. coli -derived HBV capsids or DNA and RNA ladders as described in the legend to Fig. 1 ).
Figure Legend Snippet: CsCl density gradient separation and analysis of viral particles from HepAD38 cell culture supernatant. (A) Native agarose gel analysis of viral particles. Culture supernatant of HepAD38 cells was concentrated (via ultrafiltration) and fractionated by CsCl density gradient centrifugation (3 ml of 1.18 g/cm 3 CsCl solution in the upper layer and 1.9 ml of 1.33 g/cm 3 CsCl solution in the lower layer). Viral particles in each fraction were resolved by native agarose gel electrophoresis, followed by detection of viral antigens with anti-HBsAg and anti-HBcAg antibodies and viral DNA by hybridization with minus-strand-specific riboprobe. (B to F) Southern and Northern blot detection of viral nucleic acids. Viral DNAs were separated by electrophoresis through Tris-acetate-EDTA (TAE) or alkaline (ALK) agarose gel for Southern blotting with minus- or plus-strand-specific riboprobes. Viral RNA was obtained by treatment with total nucleic acids with DNase I and separated by formaldehyde-MOPS agarose gel, followed by Northern blotting. (G) Quantification of viral DNA and RNA in naked capsids or virions. Fractions containing naked capsids (fractions 3 to 7) or virions (fractions 10 to 21) were pooled, and viral DNA and RNA were quantified by PCR. (H) DNA and RNA ratios in naked capsids and virions calculated based on quantitative results. Asterisks indicate unknown high-density viral particles detected by anti-HBcAg or anti-HBsAg antibodies but devoid of any HBV-specific nucleic acids. M, markers ( E. coli -derived HBV capsids or DNA and RNA ladders as described in the legend to Fig. 1 ).

Techniques Used: Cell Culture, Agarose Gel Electrophoresis, Gradient Centrifugation, DNA Hybridization, Northern Blot, Electrophoresis, Southern Blot, Polymerase Chain Reaction, Derivative Assay

4) Product Images from "Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist"

Article Title: Neutrophil Elastase-mediated proteolysis activates the anti-inflammatory cytokine IL-36 Receptor antagonist

Journal: Scientific Reports

doi: 10.1038/srep24880

IL-36Ra cleavage by activated PMN supernatant is prevented by serine protease inhibitors. SUMO tagged IL-36Ra was incubated with supernatant from PMNs stimulated with PMA for 1 hour at 37 °C. Incubation was performed in the presence or absence of a range of protease inhibitors. PI = complete protease inhibitor cocktail (Roche), EDTA = Ethylenediaminetetraacetic acid, inhibitor of proteases where a metal ion is required for cleavage, PI Roche = complete ultra-protease inhibitor cocktail which includes aspartic proteases (Roche), IAA = iodoacetic acid a pan cysteine protease inhibitor, PMSF = phenylmethylsulfonyl fluoride a pan serine protease inhibitor, α1 anti-trypsin is another pan serine protease inhibitor. Samples were then analysed using WB.
Figure Legend Snippet: IL-36Ra cleavage by activated PMN supernatant is prevented by serine protease inhibitors. SUMO tagged IL-36Ra was incubated with supernatant from PMNs stimulated with PMA for 1 hour at 37 °C. Incubation was performed in the presence or absence of a range of protease inhibitors. PI = complete protease inhibitor cocktail (Roche), EDTA = Ethylenediaminetetraacetic acid, inhibitor of proteases where a metal ion is required for cleavage, PI Roche = complete ultra-protease inhibitor cocktail which includes aspartic proteases (Roche), IAA = iodoacetic acid a pan cysteine protease inhibitor, PMSF = phenylmethylsulfonyl fluoride a pan serine protease inhibitor, α1 anti-trypsin is another pan serine protease inhibitor. Samples were then analysed using WB.

Techniques Used: Incubation, Protease Inhibitor, Western Blot

5) Product Images from "7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases"

Article Title: 7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks1149

DNA repair activities towards 8oxoA containing duplex oligonucleotides in extracts from E. coli and mouse cells. 2.5 nM 5′-[ 32 P]-labelled 40 mer 8oxoA•N oligonucleotide duplexes was incubated with either 30 µg of MEFs extract or 20 µg of E. coli cell extract. The repair assay (volume 100 µl) was performed in BER + EDTA buffer containing 50 mM KCl, 20 mM HEPES–KOH (pH 7.6), 0.1 mg/ml BSA, 1 mM DTT and 1 mM EDTA, for 1 h at 37°C. The reactions were stopped by adding SDS and proteinase K. ( A ) Denaturing PAGE analysis of the cleavage products after incubation of 8oxoA•N duplexes with mouse cell extracts. ( B ) Graphic representation of the mean values of cleavage activities in mouse cell extracts. ( C ) Denaturing PAGE analysis of the cleavage products after incubation of 8oxoA•N duplexes with E. coli cell extracts. ( D ) Graphic representation of the mean values of cleavage activities in E. coli cell extracts. For details see ‘Materials and Methods’ section.
Figure Legend Snippet: DNA repair activities towards 8oxoA containing duplex oligonucleotides in extracts from E. coli and mouse cells. 2.5 nM 5′-[ 32 P]-labelled 40 mer 8oxoA•N oligonucleotide duplexes was incubated with either 30 µg of MEFs extract or 20 µg of E. coli cell extract. The repair assay (volume 100 µl) was performed in BER + EDTA buffer containing 50 mM KCl, 20 mM HEPES–KOH (pH 7.6), 0.1 mg/ml BSA, 1 mM DTT and 1 mM EDTA, for 1 h at 37°C. The reactions were stopped by adding SDS and proteinase K. ( A ) Denaturing PAGE analysis of the cleavage products after incubation of 8oxoA•N duplexes with mouse cell extracts. ( B ) Graphic representation of the mean values of cleavage activities in mouse cell extracts. ( C ) Denaturing PAGE analysis of the cleavage products after incubation of 8oxoA•N duplexes with E. coli cell extracts. ( D ) Graphic representation of the mean values of cleavage activities in E. coli cell extracts. For details see ‘Materials and Methods’ section.

Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

6) Product Images from "A role for caveolin-1 in desmoglein binding and desmosome dynamics"

Article Title: A role for caveolin-1 in desmoglein binding and desmosome dynamics

Journal: Oncogene

doi: 10.1038/onc.2011.346

Localization of Dsg2 and Cav-1 to membrane lipid rafts. ( A ) A431 cells were treated with MβCD (10 mM) for 1 hr and extracted in a Tris-NaCl-EDTA buffer containing Tx. Proteins were subjected to a discontinous (5-35%) sucrose-gradient separation, resolved over SDS-PAGE and immunoblotted for Cav-1, Cav-2, Flo1, Flo2, β-Cat, γ-Cat, actin, E-Cad and Dsg2. Immunoblotting revealed that Cav-1 localized predominantly to low-density fractions 4 and 5 (top left panel), corresponding to lipid rafts. Dsg2 was distributed through all fractions from 4-12. Treatment with MβCD (10 mM) for 1 hr, disrupted lipid rafts, and shifted both Cav-1 and Dsg2 to the more dense fractions. In addition to the 160 kDa Dsg2 full-length protein, we observed a 65 kDa band in the lipid raft fraction # 4 (vertical arrow). Accumulation of this fragment was enhanced and shifted to the denser fractions in the presence of MβCD (arrow head). We note that β-Cat, γ-Cat, E-Cad and actin fractioned to the lower, denser fractions, and remained relatively unchanged in the presence of MβCD. ( B ) Proteins from fractions 4 (lipid raft fraction) and 12 (high molecular weight, non-raft fraction) from above were resolved over SDS-PAGE and immunoblotted for Dsg2 using two different antibodies, 10D2 and DG3.10. Treatment with MβCD increased the level of the 65-kDa Dsg2 fragment as detected by DG3.10, but not 10D2.
Figure Legend Snippet: Localization of Dsg2 and Cav-1 to membrane lipid rafts. ( A ) A431 cells were treated with MβCD (10 mM) for 1 hr and extracted in a Tris-NaCl-EDTA buffer containing Tx. Proteins were subjected to a discontinous (5-35%) sucrose-gradient separation, resolved over SDS-PAGE and immunoblotted for Cav-1, Cav-2, Flo1, Flo2, β-Cat, γ-Cat, actin, E-Cad and Dsg2. Immunoblotting revealed that Cav-1 localized predominantly to low-density fractions 4 and 5 (top left panel), corresponding to lipid rafts. Dsg2 was distributed through all fractions from 4-12. Treatment with MβCD (10 mM) for 1 hr, disrupted lipid rafts, and shifted both Cav-1 and Dsg2 to the more dense fractions. In addition to the 160 kDa Dsg2 full-length protein, we observed a 65 kDa band in the lipid raft fraction # 4 (vertical arrow). Accumulation of this fragment was enhanced and shifted to the denser fractions in the presence of MβCD (arrow head). We note that β-Cat, γ-Cat, E-Cad and actin fractioned to the lower, denser fractions, and remained relatively unchanged in the presence of MβCD. ( B ) Proteins from fractions 4 (lipid raft fraction) and 12 (high molecular weight, non-raft fraction) from above were resolved over SDS-PAGE and immunoblotted for Dsg2 using two different antibodies, 10D2 and DG3.10. Treatment with MβCD increased the level of the 65-kDa Dsg2 fragment as detected by DG3.10, but not 10D2.

Techniques Used: SDS Page, Molecular Weight

7) Product Images from "Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli"

Article Title: Crystal Structure of Penicillin-Binding Protein 3 (PBP3) from Escherichia coli

Journal: PLoS ONE

doi: 10.1371/journal.pone.0098042

PBP3 oligomerization. (a) Chromatogram of PBP3 88–165 gel filtration on a Superdex 75 10/300 GL. The first peak elutes at 12.16 ml and the second at 13.52 ml. Carbonic anhydrase (31 kDa) elutes at 11.05 ml and lysozyme (14 kDa) at 15.25 ml (data not shown). The buffer was 0.15 M NaCl and 0.1 M Tris, pH 8 1 mM EDTA. (b) Chromatogram of PBP3 57–577 gel filtration on a Superdex 200 10/300 GL. The first small peak elutes at 13.3 ml, the second at 14.77 ml. Bovine serum albumin used as a standard elutes at 14.12 ml (molecular mass 67 kDa, data not shown). The masses calculated on the basis of the mass standards are 108.5 kDa for the first peak (PBP3 57–577 dimer) and 58.5 kDa for the second peak (PBP3 57–577 monomer). The buffer was 20 mM Tris HCl pH 8, 0.5 M NaCl.
Figure Legend Snippet: PBP3 oligomerization. (a) Chromatogram of PBP3 88–165 gel filtration on a Superdex 75 10/300 GL. The first peak elutes at 12.16 ml and the second at 13.52 ml. Carbonic anhydrase (31 kDa) elutes at 11.05 ml and lysozyme (14 kDa) at 15.25 ml (data not shown). The buffer was 0.15 M NaCl and 0.1 M Tris, pH 8 1 mM EDTA. (b) Chromatogram of PBP3 57–577 gel filtration on a Superdex 200 10/300 GL. The first small peak elutes at 13.3 ml, the second at 14.77 ml. Bovine serum albumin used as a standard elutes at 14.12 ml (molecular mass 67 kDa, data not shown). The masses calculated on the basis of the mass standards are 108.5 kDa for the first peak (PBP3 57–577 dimer) and 58.5 kDa for the second peak (PBP3 57–577 monomer). The buffer was 20 mM Tris HCl pH 8, 0.5 M NaCl.

Techniques Used: Filtration

8) Product Images from "The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin"

Article Title: The N-terminal 70-kDa fragment of fibronectin binds to cell surface fibronectin assembly sites in the absence of intact fibronectin

Journal: Matrix biology : journal of the International Society for Matrix Biology

doi: 10.1016/j.matbio.2006.02.002

Cell-bound FITC–70K does not form detergent-insoluble multimers. FN –/– cells were plated on laminin-coated tissue culture plastic for 2h. 40nM FITC–70K or 20nM FITC–FN were added for 10min, 1h or 24h in 400nM LPA, DMEM and 0.2% BSA. Monolayers were washed and extracted with 1% deoxycholate in TBS, 2mM EDTA, 2mM iodoacetamide and proteinase inhibitor complex. Samples were centrifuged to separate insoluble from soluble fractions. Samples were run unreduced on 8% acrylamide gels and immunoblotted with rabbit anti-FITC antibodies, followed by peroxidase anti-rabbit conjugates and chemiluminescence development. Lanes: (1) FITC–70K, (2) FITC–FN. Migration of molecular weight standards (kDa) is depicted to the left. The arrow indicates the well level at the top of the 3.5% stacking gel.
Figure Legend Snippet: Cell-bound FITC–70K does not form detergent-insoluble multimers. FN –/– cells were plated on laminin-coated tissue culture plastic for 2h. 40nM FITC–70K or 20nM FITC–FN were added for 10min, 1h or 24h in 400nM LPA, DMEM and 0.2% BSA. Monolayers were washed and extracted with 1% deoxycholate in TBS, 2mM EDTA, 2mM iodoacetamide and proteinase inhibitor complex. Samples were centrifuged to separate insoluble from soluble fractions. Samples were run unreduced on 8% acrylamide gels and immunoblotted with rabbit anti-FITC antibodies, followed by peroxidase anti-rabbit conjugates and chemiluminescence development. Lanes: (1) FITC–70K, (2) FITC–FN. Migration of molecular weight standards (kDa) is depicted to the left. The arrow indicates the well level at the top of the 3.5% stacking gel.

Techniques Used: Migration, Molecular Weight

9) Product Images from "Comparison of Blood Collected in Acid-Citrate-Dextrose and EDTA for Use in Human Immunodeficiency Virus Peripheral Blood Mononuclear Cell Cultures"

Article Title: Comparison of Blood Collected in Acid-Citrate-Dextrose and EDTA for Use in Human Immunodeficiency Virus Peripheral Blood Mononuclear Cell Cultures

Journal: Journal of Clinical Microbiology

doi:

Sensitivity of HIV PBMC cultures according to log 10 HIV RNA plasma viral loads with ACD or EDTA as the anticoagulant.
Figure Legend Snippet: Sensitivity of HIV PBMC cultures according to log 10 HIV RNA plasma viral loads with ACD or EDTA as the anticoagulant.

Techniques Used:

10) Product Images from "Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)"

Article Title: Activation of MAPK/ERK signaling by Burkholderia pseudomallei cycle inhibiting factor (Cif)

Journal: PLoS ONE

doi: 10.1371/journal.pone.0171464

Cif expression regulates the CDC25-H and proline-rich domains in SOS1. (A,B) HEK293T cells were transfected with the indicated expression plasmids for two days, followed by FLAG immunoprecipitation of the cell lysates using FLAG-agarose and Western blotting of the immunoprecipitates and aliquots of the cell lysates using the indicated antibodies. (C) All cells were transfected with FLAG-SOS1 as well as V5-Cif or empty vector, as indicated, for two days. Cells were then lysed in hypotonic lysis buffer (25 mM Tris-HCL, 2 mM EDTA, 2 mM EGTA, 0.1% β-Mercaptoethanol, Roche protease inhibitor cocktail, pH 7.5) and cell lysates subjected to freeze-thawing at -80°C. The cytosolic fraction was separated from the remaining cellular compartments (membranes and nuclear fraction) through centrifugation. The proteins in the cytosolic supernatant and the pellet were denatured using SDS loading buffer and analysed using Western blotting with the indicated antibodies. Equal corresponding amounts of cytosol and pellet were loaded. GAPDH served as a control for the cytosolic fraction and calnexin as a control for the membrane fraction. Glut1 (55 kda) served as an additional control for the membrane fraction, as the higher molecular weight, glycosylated Glut1 species can only be detected in the membrane fraction. (D) Cells were transfected with the indicated FLAG-mSOS1 truncation constructs for two days, followed by Western blotting using the indicated antibodies. The bottom FLAG blot in the Western blot panel was obtained from running a duplicate set of lysates through another SDS-PAGE gel of a lower resolving gel percentage for a longer time to increase the resolving power.
Figure Legend Snippet: Cif expression regulates the CDC25-H and proline-rich domains in SOS1. (A,B) HEK293T cells were transfected with the indicated expression plasmids for two days, followed by FLAG immunoprecipitation of the cell lysates using FLAG-agarose and Western blotting of the immunoprecipitates and aliquots of the cell lysates using the indicated antibodies. (C) All cells were transfected with FLAG-SOS1 as well as V5-Cif or empty vector, as indicated, for two days. Cells were then lysed in hypotonic lysis buffer (25 mM Tris-HCL, 2 mM EDTA, 2 mM EGTA, 0.1% β-Mercaptoethanol, Roche protease inhibitor cocktail, pH 7.5) and cell lysates subjected to freeze-thawing at -80°C. The cytosolic fraction was separated from the remaining cellular compartments (membranes and nuclear fraction) through centrifugation. The proteins in the cytosolic supernatant and the pellet were denatured using SDS loading buffer and analysed using Western blotting with the indicated antibodies. Equal corresponding amounts of cytosol and pellet were loaded. GAPDH served as a control for the cytosolic fraction and calnexin as a control for the membrane fraction. Glut1 (55 kda) served as an additional control for the membrane fraction, as the higher molecular weight, glycosylated Glut1 species can only be detected in the membrane fraction. (D) Cells were transfected with the indicated FLAG-mSOS1 truncation constructs for two days, followed by Western blotting using the indicated antibodies. The bottom FLAG blot in the Western blot panel was obtained from running a duplicate set of lysates through another SDS-PAGE gel of a lower resolving gel percentage for a longer time to increase the resolving power.

Techniques Used: Expressing, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Lysis, Protease Inhibitor, Centrifugation, Molecular Weight, Construct, SDS Page

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Centrifugation:

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Mass Spectrometry:

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Construct:

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Incubation:

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Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue
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Article Title: Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
Article Snippet: The bacteria were harvested by centrifugation at 3,300 g at 4 °C for 1 h. Cell pellets were resuspended in 100 ml of lysis buffer containing 20 mM Tris HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP pH8, 15% glycerol and 2 tablets of Complete ultra protease inhibitors without EDTA (Roche). .. For the purification of the recombinant proteins, the bacterial suspension was incubated with DNAase (30 μg) on ice for 15 min, homogenized in a Microfluid instrument (Model M-110 P) and centrifuged at 33,000 g at 4 °C for 1 h. The GST-tagged recombinant proteins were purified using GSTrap HP chromatography (GE Healthcare Life Sciences).

Expressing:

Article Title: Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
Article Snippet: E. coli BL21 (DE3) pLysS cells (Invitrogen) transformed with pGEX-6P-1-CLIC3 vector were grown at 37 °C until the cell density reached an OD600 of 0.6, at which point GST-CLIC3/CLIC3C22A expression was induced with 0.25 mM isopropyl β-D -thiogalactosidase (IPTG) at 30 °C for 2 h. The bacteria were harvested by centrifugation at 3,300 g at 4 °C for 1 h. Cell pellets were resuspended in 100 ml of lysis buffer containing 0.1% Triton X-100, 2 mM Benzamidine, 3 μM Pepstatin, 3 μM Antipain, 4 μM Leupeptin and 0.3 μM Aprotinin in PBS pH 7.4. .. The bacteria were harvested by centrifugation at 3,300 g at 4 °C for 1 h. Cell pellets were resuspended in 100 ml of lysis buffer containing 20 mM Tris HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP pH8, 15% glycerol and 2 tablets of Complete ultra protease inhibitors without EDTA (Roche).

Western Blot:

Article Title: Heterotypic signals from neural HSF-1 separate thermotolerance from longevity
Article Snippet: Paragraph title: Western Blot Analysis ... The supernatant was supplemented with 2x SDS sample buffer containing [50mM Tris-Cl at pH 6.8, 2 mM EDTA, 4% glycerol, 2% SDS, Coomassie Blue, protease inhibitor cocktail without EDTA (Roche)].

Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue
Article Snippet: .. A standard lysis buffer formulation was selected due to the authors’ familiarity with it, which has been used for direct homogenization and Western blotting in work done previously by : 1mM EDTA, 140mM NaCl, 2% SDS, 10mM Tris pH 8.0, 50mM NaF, 1mM activated NaOv, protease inhibitor table (1 tablet per 50mL solution; Roche). ..

Article Title: Aging Reduces the Activation of the mTORC1 Pathway after Resistance Exercise and Protein Intake in Human Skeletal Muscle: Potential Role of REDD1 and Impaired Anabolic Sensitivity
Article Snippet: .. Western Blotting Frozen muscle samples were homogenized in a buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM fluoride, 1 mM DTT (1,4-dithiothreitol), and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science, Vilvoorde, Belgium). .. Protein concentration was determined using the DC protein assay kit (Biorad, Nazareth Eke, Belgium) with bovine serum albumin as a standard.

Article Title: The chaperone-like protein CDC48 regulates ascorbate peroxidase in tobacco
Article Snippet: Immunoblotting Proteins from tobacco cells were quantified using the Bradford method ( ) after disruption in lysis buffer, which consisted of 50 mM HEPES, pH 7.5, 50 mM EDTA, 2 mM dithiothreitol (DTT), 100 mM NaCl, and protease inhibitor cocktail (PIC) without EDTA (Roche), either supplemented or not with 10 mM N-Ethylmaleimide (NEM). .. The immunoblots were examined using LumiGLO® (Cell Signaling Technology).

Article Title: Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components
Article Snippet: Paragraph title: Western blotting ... For high-salt buffer protein extraction experiments, the following buffers have been tried: (i) 750 mM NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM NaF, 5 mM EDTA; (ii) 750 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, proteinase inhibitor cocktail (Roche), 0.1% Triton X-100; (iii) 5% SDS, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 175 mM NaCl; (iv) 5% SDS, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 175 mM NaCl, 8 M urea; (v) 750 mM NaCl, 50 mM Tris-HCl (pH 8.8), 10 mM NaF, 5 mM EDTA; (vi) 750 mM NaCl, 50 mM Tris, 10 mM NaF, 5 mM EDTA; and (vi) Bio-Rad ReadyPrep Protein Extraction Kit (Soluble/Insoluble, 163-2083).

Transformation Assay:

Article Title: Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
Article Snippet: For GST-TGM2 construct, E. coli BL21 (DE3) pLysS cells (Invitrogen) transformed with pGEX-6P-1-TGM2 vector (kindly provided by Professor Jeffrey Keillor) were grown at 25 °C until the cell density reached an OD600 reading of 0.6, at which point the temperature was reduced to 18 °C before overnight induction with 1 μM IPTG. .. The bacteria were harvested by centrifugation at 3,300 g at 4 °C for 1 h. Cell pellets were resuspended in 100 ml of lysis buffer containing 20 mM Tris HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP pH8, 15% glycerol and 2 tablets of Complete ultra protease inhibitors without EDTA (Roche).

Chromatography:

Article Title: Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
Article Snippet: The bacteria were harvested by centrifugation at 3,300 g at 4 °C for 1 h. Cell pellets were resuspended in 100 ml of lysis buffer containing 20 mM Tris HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP pH8, 15% glycerol and 2 tablets of Complete ultra protease inhibitors without EDTA (Roche). .. For the purification of the recombinant proteins, the bacterial suspension was incubated with DNAase (30 μg) on ice for 15 min, homogenized in a Microfluid instrument (Model M-110 P) and centrifuged at 33,000 g at 4 °C for 1 h. The GST-tagged recombinant proteins were purified using GSTrap HP chromatography (GE Healthcare Life Sciences).

Protease Inhibitor:

Article Title: Heterotypic signals from neural HSF-1 separate thermotolerance from longevity
Article Snippet: .. The supernatant was supplemented with 2x SDS sample buffer containing [50mM Tris-Cl at pH 6.8, 2 mM EDTA, 4% glycerol, 2% SDS, Coomassie Blue, protease inhibitor cocktail without EDTA (Roche)]. ..

Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue
Article Snippet: .. A standard lysis buffer formulation was selected due to the authors’ familiarity with it, which has been used for direct homogenization and Western blotting in work done previously by : 1mM EDTA, 140mM NaCl, 2% SDS, 10mM Tris pH 8.0, 50mM NaF, 1mM activated NaOv, protease inhibitor table (1 tablet per 50mL solution; Roche). ..

Article Title: The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability
Article Snippet: .. Protein extraction was performed by bead beating in buffer A (40 mM Hepes-NaOH [pH 7.5], 120 mM NaCl, 1 mM EDTA, 0.3% CHAPS, 50 mM NaF, 10 mM β-glycerophosphate) supplemented with complete protease inhibitor tablets without EDTA (Roche). .. Extracts were cleared by centrifugation and incubated overnight at 4°C with anti-Vma1 or anti-Vph1 (Abcam, 8B1 and 10D7).

Article Title: Aging Reduces the Activation of the mTORC1 Pathway after Resistance Exercise and Protein Intake in Human Skeletal Muscle: Potential Role of REDD1 and Impaired Anabolic Sensitivity
Article Snippet: .. Western Blotting Frozen muscle samples were homogenized in a buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM fluoride, 1 mM DTT (1,4-dithiothreitol), and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science, Vilvoorde, Belgium). .. Protein concentration was determined using the DC protein assay kit (Biorad, Nazareth Eke, Belgium) with bovine serum albumin as a standard.

Article Title: Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions
Article Snippet: .. Cells were scraped in DPBS++ containing protease inhibitor cocktail without EDTA (Roche) and centrifuged at 4 °C, 500 g for 8 min. Next, cells were resuspended in DPBS++ with protease inhibitor cocktail without EDTA (Roche) containing 0.1% (v/v) Triton X-100, sonicated 3 × for 1 s and incubated for 30 min on ice. ..

Article Title: Regulation of ubiquitin-proteasome and autophagy pathways after acute LPS and epoxomicin administration in mice
Article Snippet: .. The other part was homogenized in ice cold buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM DTT (1,4-dithiothreitol) and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science, Vilvoorde, Belgium). .. Protein content was determined using the DC protein assay kit (Bio-Rad, Nazareth Eke, Belgium) with bovine serum albumin (BSA) as a standard.

Article Title: The chaperone-like protein CDC48 regulates ascorbate peroxidase in tobacco
Article Snippet: .. Immunoblotting Proteins from tobacco cells were quantified using the Bradford method ( ) after disruption in lysis buffer, which consisted of 50 mM HEPES, pH 7.5, 50 mM EDTA, 2 mM dithiothreitol (DTT), 100 mM NaCl, and protease inhibitor cocktail (PIC) without EDTA (Roche), either supplemented or not with 10 mM N-Ethylmaleimide (NEM). .. Protein samples (20 μg) were resolved by 10–15% SDS-PAGE or 6% native-PAGE and visualized by immunoblotting with antibodies against CDC48 (Abcam), cAPX (Agrisera), or His-tag (Cell Signaling Technology).

Cell Culture:

Article Title: Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions
Article Snippet: Co-immunoprecipitation AECs were isolated and 2.5 × 106 cells per well were plated on six-well Transwell-permeable supports (Corning 3450) coated with 20 μg ml−1 rat tail collagen (Roche, Nutley, NJ) and cultured for 6 days as described above. .. Cells were scraped in DPBS++ containing protease inhibitor cocktail without EDTA (Roche) and centrifuged at 4 °C, 500 g for 8 min. Next, cells were resuspended in DPBS++ with protease inhibitor cocktail without EDTA (Roche) containing 0.1% (v/v) Triton X-100, sonicated 3 × for 1 s and incubated for 30 min on ice.

Generated:

Article Title: Heterotypic signals from neural HSF-1 separate thermotolerance from longevity
Article Snippet: Worm extracts were generated by glass bead disruption in non-denaturing lysis buffer [150mM NaCl, 50mM Hepes at pH 7.4, 1mM EDTA, 1% Triton X100, protease inhibitor cocktail without EDTA (Roche)]. .. The supernatant was supplemented with 2x SDS sample buffer containing [50mM Tris-Cl at pH 6.8, 2 mM EDTA, 4% glycerol, 2% SDS, Coomassie Blue, protease inhibitor cocktail without EDTA (Roche)].

Inhibition:

Article Title: The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability
Article Snippet: For V-ATPase assembly analysis by sch9 as inhibition, precultures were diluted in YPD medium (pH 5, 50 mM MES) with or without 300 nm 1-NM-PP1 (Merck-Millipore) and grown for 6 hours, after which cultures were starved for glucose in the absence or presence of the inhibitor. .. Protein extraction was performed by bead beating in buffer A (40 mM Hepes-NaOH [pH 7.5], 120 mM NaCl, 1 mM EDTA, 0.3% CHAPS, 50 mM NaF, 10 mM β-glycerophosphate) supplemented with complete protease inhibitor tablets without EDTA (Roche).

Protein Concentration:

Article Title: Aging Reduces the Activation of the mTORC1 Pathway after Resistance Exercise and Protein Intake in Human Skeletal Muscle: Potential Role of REDD1 and Impaired Anabolic Sensitivity
Article Snippet: Western Blotting Frozen muscle samples were homogenized in a buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM fluoride, 1 mM DTT (1,4-dithiothreitol), and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science, Vilvoorde, Belgium). .. Protein concentration was determined using the DC protein assay kit (Biorad, Nazareth Eke, Belgium) with bovine serum albumin as a standard.

Sonication:

Article Title: Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions
Article Snippet: .. Cells were scraped in DPBS++ containing protease inhibitor cocktail without EDTA (Roche) and centrifuged at 4 °C, 500 g for 8 min. Next, cells were resuspended in DPBS++ with protease inhibitor cocktail without EDTA (Roche) containing 0.1% (v/v) Triton X-100, sonicated 3 × for 1 s and incubated for 30 min on ice. ..

Recombinant:

Article Title: Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
Article Snippet: Paragraph title: Recombinant proteins generation and purification ... The bacteria were harvested by centrifugation at 3,300 g at 4 °C for 1 h. Cell pellets were resuspended in 100 ml of lysis buffer containing 20 mM Tris HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP pH8, 15% glycerol and 2 tablets of Complete ultra protease inhibitors without EDTA (Roche).

DC Protein Assay:

Article Title: Aging Reduces the Activation of the mTORC1 Pathway after Resistance Exercise and Protein Intake in Human Skeletal Muscle: Potential Role of REDD1 and Impaired Anabolic Sensitivity
Article Snippet: Western Blotting Frozen muscle samples were homogenized in a buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM fluoride, 1 mM DTT (1,4-dithiothreitol), and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science, Vilvoorde, Belgium). .. Protein concentration was determined using the DC protein assay kit (Biorad, Nazareth Eke, Belgium) with bovine serum albumin as a standard.

Article Title: Regulation of ubiquitin-proteasome and autophagy pathways after acute LPS and epoxomicin administration in mice
Article Snippet: The other part was homogenized in ice cold buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM DTT (1,4-dithiothreitol) and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science, Vilvoorde, Belgium). .. Protein content was determined using the DC protein assay kit (Bio-Rad, Nazareth Eke, Belgium) with bovine serum albumin (BSA) as a standard.

Magnetic Beads:

Article Title: Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions
Article Snippet: Cells were scraped in DPBS++ containing protease inhibitor cocktail without EDTA (Roche) and centrifuged at 4 °C, 500 g for 8 min. Next, cells were resuspended in DPBS++ with protease inhibitor cocktail without EDTA (Roche) containing 0.1% (v/v) Triton X-100, sonicated 3 × for 1 s and incubated for 30 min on ice. .. Before use, protein A magnetic beads (Sure Beads; BioRad) for co-immunoprecipitation were washed 3 × in DPBS++ (100 μl beads per 1 ml) and then blocked with DPBS++ containing protease inhibitor cocktail, 0.25% BSA and 0.2% Gelatin for 1 h at 4 °C.

Isolation:

Article Title: Regulation of claudin/zonula occludens-1 complexes by hetero-claudin interactions
Article Snippet: Co-immunoprecipitation AECs were isolated and 2.5 × 106 cells per well were plated on six-well Transwell-permeable supports (Corning 3450) coated with 20 μg ml−1 rat tail collagen (Roche, Nutley, NJ) and cultured for 6 days as described above. .. Cells were scraped in DPBS++ containing protease inhibitor cocktail without EDTA (Roche) and centrifuged at 4 °C, 500 g for 8 min. Next, cells were resuspended in DPBS++ with protease inhibitor cocktail without EDTA (Roche) containing 0.1% (v/v) Triton X-100, sonicated 3 × for 1 s and incubated for 30 min on ice.

Size-exclusion Chromatography:

Article Title: Heterotypic signals from neural HSF-1 separate thermotolerance from longevity
Article Snippet: Worms were centrifuged at 1000 × g for 30 sec and move back to NG plates seeded with OP50 bacteria at 20°C. .. The supernatant was supplemented with 2x SDS sample buffer containing [50mM Tris-Cl at pH 6.8, 2 mM EDTA, 4% glycerol, 2% SDS, Coomassie Blue, protease inhibitor cocktail without EDTA (Roche)].

Purification:

Article Title: Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
Article Snippet: Paragraph title: Recombinant proteins generation and purification ... The bacteria were harvested by centrifugation at 3,300 g at 4 °C for 1 h. Cell pellets were resuspended in 100 ml of lysis buffer containing 20 mM Tris HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP pH8, 15% glycerol and 2 tablets of Complete ultra protease inhibitors without EDTA (Roche).

Protein Extraction:

Article Title: The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability
Article Snippet: .. Protein extraction was performed by bead beating in buffer A (40 mM Hepes-NaOH [pH 7.5], 120 mM NaCl, 1 mM EDTA, 0.3% CHAPS, 50 mM NaF, 10 mM β-glycerophosphate) supplemented with complete protease inhibitor tablets without EDTA (Roche). .. Extracts were cleared by centrifugation and incubated overnight at 4°C with anti-Vma1 or anti-Vph1 (Abcam, 8B1 and 10D7).

Article Title: Regulation of ubiquitin-proteasome and autophagy pathways after acute LPS and epoxomicin administration in mice
Article Snippet: Paragraph title: Protein extraction for immunoblotting ... The other part was homogenized in ice cold buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM DTT (1,4-dithiothreitol) and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science, Vilvoorde, Belgium).

Article Title: Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components
Article Snippet: .. For high-salt buffer protein extraction experiments, the following buffers have been tried: (i) 750 mM NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM NaF, 5 mM EDTA; (ii) 750 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, proteinase inhibitor cocktail (Roche), 0.1% Triton X-100; (iii) 5% SDS, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 175 mM NaCl; (iv) 5% SDS, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 175 mM NaCl, 8 M urea; (v) 750 mM NaCl, 50 mM Tris-HCl (pH 8.8), 10 mM NaF, 5 mM EDTA; (vi) 750 mM NaCl, 50 mM Tris, 10 mM NaF, 5 mM EDTA; and (vi) Bio-Rad ReadyPrep Protein Extraction Kit (Soluble/Insoluble, 163-2083). ..

Blocking Assay:

Article Title: Aging Reduces the Activation of the mTORC1 Pathway after Resistance Exercise and Protein Intake in Human Skeletal Muscle: Potential Role of REDD1 and Impaired Anabolic Sensitivity
Article Snippet: Western Blotting Frozen muscle samples were homogenized in a buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM fluoride, 1 mM DTT (1,4-dithiothreitol), and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science, Vilvoorde, Belgium). .. After a blocking step of 1 h, membranes were probed with the following primary antibodies overnight: phospho-Thr389 S6K1, phospho-Ser2448 mTOR, phospho-Thr246 proline-rich Akt substrate of 40 kDa (PRAS40), phospho-Ser473 Akt/protein kinase B (Akt/PKB), phospho-Thr308 Akt/PKB, phospho-Thr172 AMPK, phospho-Ser79 Acetyl CoA carboxylase (ACC), LC3b, hypoxia-inducible factor 1 alpha (HIF-1α), eukaryotic initiation factor 2 (eEF2) (Cell Signaling, Leiden, The Netherlands), p62 (Progen, Heidelberg, Germany), and REDD1 (ProteinTech, Manchester, UK).

Polyacrylamide Gel Electrophoresis:

Article Title: Aging Reduces the Activation of the mTORC1 Pathway after Resistance Exercise and Protein Intake in Human Skeletal Muscle: Potential Role of REDD1 and Impaired Anabolic Sensitivity
Article Snippet: Western Blotting Frozen muscle samples were homogenized in a buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM fluoride, 1 mM DTT (1,4-dithiothreitol), and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science, Vilvoorde, Belgium). .. For immunoblotting, 30 µg of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinlidene difluoride (PVDF) membrane.

Lysis:

Article Title: Heterotypic signals from neural HSF-1 separate thermotolerance from longevity
Article Snippet: Worm extracts were generated by glass bead disruption in non-denaturing lysis buffer [150mM NaCl, 50mM Hepes at pH 7.4, 1mM EDTA, 1% Triton X100, protease inhibitor cocktail without EDTA (Roche)]. .. The supernatant was supplemented with 2x SDS sample buffer containing [50mM Tris-Cl at pH 6.8, 2 mM EDTA, 4% glycerol, 2% SDS, Coomassie Blue, protease inhibitor cocktail without EDTA (Roche)].

Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue
Article Snippet: .. A standard lysis buffer formulation was selected due to the authors’ familiarity with it, which has been used for direct homogenization and Western blotting in work done previously by : 1mM EDTA, 140mM NaCl, 2% SDS, 10mM Tris pH 8.0, 50mM NaF, 1mM activated NaOv, protease inhibitor table (1 tablet per 50mL solution; Roche). ..

Article Title: Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
Article Snippet: .. The bacteria were harvested by centrifugation at 3,300 g at 4 °C for 1 h. Cell pellets were resuspended in 100 ml of lysis buffer containing 20 mM Tris HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP pH8, 15% glycerol and 2 tablets of Complete ultra protease inhibitors without EDTA (Roche). .. For the purification of the recombinant proteins, the bacterial suspension was incubated with DNAase (30 μg) on ice for 15 min, homogenized in a Microfluid instrument (Model M-110 P) and centrifuged at 33,000 g at 4 °C for 1 h. The GST-tagged recombinant proteins were purified using GSTrap HP chromatography (GE Healthcare Life Sciences).

Article Title: The chaperone-like protein CDC48 regulates ascorbate peroxidase in tobacco
Article Snippet: .. Immunoblotting Proteins from tobacco cells were quantified using the Bradford method ( ) after disruption in lysis buffer, which consisted of 50 mM HEPES, pH 7.5, 50 mM EDTA, 2 mM dithiothreitol (DTT), 100 mM NaCl, and protease inhibitor cocktail (PIC) without EDTA (Roche), either supplemented or not with 10 mM N-Ethylmaleimide (NEM). .. Protein samples (20 μg) were resolved by 10–15% SDS-PAGE or 6% native-PAGE and visualized by immunoblotting with antibodies against CDC48 (Abcam), cAPX (Agrisera), or His-tag (Cell Signaling Technology).

SDS Page:

Article Title: Aging Reduces the Activation of the mTORC1 Pathway after Resistance Exercise and Protein Intake in Human Skeletal Muscle: Potential Role of REDD1 and Impaired Anabolic Sensitivity
Article Snippet: Western Blotting Frozen muscle samples were homogenized in a buffer containing 20 mM Tris, pH 7.0, 270 mM sucrose, 5 mM EGTA, 1 mM EDTA, 1% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium β-glycerophosphate, 5 mM sodium pyrophosphate, 50 mM fluoride, 1 mM DTT (1,4-dithiothreitol), and a protease inhibitor cocktail containing 1 mM EDTA (Roche Applied Science, Vilvoorde, Belgium). .. For immunoblotting, 30 µg of proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinlidene difluoride (PVDF) membrane.

Article Title: The chaperone-like protein CDC48 regulates ascorbate peroxidase in tobacco
Article Snippet: Immunoblotting Proteins from tobacco cells were quantified using the Bradford method ( ) after disruption in lysis buffer, which consisted of 50 mM HEPES, pH 7.5, 50 mM EDTA, 2 mM dithiothreitol (DTT), 100 mM NaCl, and protease inhibitor cocktail (PIC) without EDTA (Roche), either supplemented or not with 10 mM N-Ethylmaleimide (NEM). .. Protein samples (20 μg) were resolved by 10–15% SDS-PAGE or 6% native-PAGE and visualized by immunoblotting with antibodies against CDC48 (Abcam), cAPX (Agrisera), or His-tag (Cell Signaling Technology).

Plasmid Preparation:

Article Title: Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
Article Snippet: For GST-TGM2 construct, E. coli BL21 (DE3) pLysS cells (Invitrogen) transformed with pGEX-6P-1-TGM2 vector (kindly provided by Professor Jeffrey Keillor) were grown at 25 °C until the cell density reached an OD600 reading of 0.6, at which point the temperature was reduced to 18 °C before overnight induction with 1 μM IPTG. .. The bacteria were harvested by centrifugation at 3,300 g at 4 °C for 1 h. Cell pellets were resuspended in 100 ml of lysis buffer containing 20 mM Tris HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP pH8, 15% glycerol and 2 tablets of Complete ultra protease inhibitors without EDTA (Roche).

In Situ:

Article Title: Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity
Article Snippet: The bacteria were harvested by centrifugation at 3,300 g at 4 °C for 1 h. Cell pellets were resuspended in 100 ml of lysis buffer containing 20 mM Tris HCl pH 8, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP pH8, 15% glycerol and 2 tablets of Complete ultra protease inhibitors without EDTA (Roche). .. The GST was cleaved from the purified proteins in situ by proteolysis with PreScission protease (Life Technologies) according to the manufacturer's instructions, and the recombinant GST-free proteins eluted, concentrated (Amicon ultra 15, 10 kDa, Millipore) and stored at −80 °C until use.

Homogenization:

Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue
Article Snippet: .. A standard lysis buffer formulation was selected due to the authors’ familiarity with it, which has been used for direct homogenization and Western blotting in work done previously by : 1mM EDTA, 140mM NaCl, 2% SDS, 10mM Tris pH 8.0, 50mM NaF, 1mM activated NaOv, protease inhibitor table (1 tablet per 50mL solution; Roche). ..

Produced:

Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue
Article Snippet: A standard lysis buffer formulation was selected due to the authors’ familiarity with it, which has been used for direct homogenization and Western blotting in work done previously by : 1mM EDTA, 140mM NaCl, 2% SDS, 10mM Tris pH 8.0, 50mM NaF, 1mM activated NaOv, protease inhibitor table (1 tablet per 50mL solution; Roche). .. To determine if simultaneously varying the concentrations of these chemicals could further enhance the solubilization process, we formulated an ‘optimized’ lysis buffer, defined as the combination of chemical concentrations that produced the nominally highest protein yields in (20mM EDTA, 140mM NaCl, 5% SDS, 100mM Tris pH 8.0).

Concentration Assay:

Article Title: Optimized solubilization of TRIzol-precipitated protein permits Western blotting analysis to maximize data available from brain tissue
Article Snippet: A standard lysis buffer formulation was selected due to the authors’ familiarity with it, which has been used for direct homogenization and Western blotting in work done previously by : 1mM EDTA, 140mM NaCl, 2% SDS, 10mM Tris pH 8.0, 50mM NaF, 1mM activated NaOv, protease inhibitor table (1 tablet per 50mL solution; Roche). .. Because the initial concentration gradients that revealed the effect of concentration on solubilization ( ) covered a broad range, we next determined if smaller changes in concentration around the optimized level would further improve protein yield.

Staining:

Article Title: The chaperone-like protein CDC48 regulates ascorbate peroxidase in tobacco
Article Snippet: Immunoblotting Proteins from tobacco cells were quantified using the Bradford method ( ) after disruption in lysis buffer, which consisted of 50 mM HEPES, pH 7.5, 50 mM EDTA, 2 mM dithiothreitol (DTT), 100 mM NaCl, and protease inhibitor cocktail (PIC) without EDTA (Roche), either supplemented or not with 10 mM N-Ethylmaleimide (NEM). .. After transfer, membranes were stained with Ponceau Red in order to check the loading of total proteins.

Clear Native PAGE:

Article Title: The chaperone-like protein CDC48 regulates ascorbate peroxidase in tobacco
Article Snippet: Immunoblotting Proteins from tobacco cells were quantified using the Bradford method ( ) after disruption in lysis buffer, which consisted of 50 mM HEPES, pH 7.5, 50 mM EDTA, 2 mM dithiothreitol (DTT), 100 mM NaCl, and protease inhibitor cocktail (PIC) without EDTA (Roche), either supplemented or not with 10 mM N-Ethylmaleimide (NEM). .. Protein samples (20 μg) were resolved by 10–15% SDS-PAGE or 6% native-PAGE and visualized by immunoblotting with antibodies against CDC48 (Abcam), cAPX (Agrisera), or His-tag (Cell Signaling Technology).

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    Roche n m edta
    N M Edta, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/n m edta/product/Roche
    Average 91 stars, based on 4 article reviews
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    edta  (Roche)
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    Quantitative <t>PCR.</t> Total RNA extracted from tibiae decalicified with 0.5 M <t>EDTA,</t> RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.
    Edta, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 524 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/edta/product/Roche
    Average 99 stars, based on 524 article reviews
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    Quantitative <t>PCR.</t> Total RNA extracted from tibiae decalicified with 0.5 M <t>EDTA,</t> RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.
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    Quantitative PCR. Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Quantitative PCR. Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, SYBR Green Assay, Fluorescence

    Fluorescent immunohistochemistry of articular cartilage. Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA later /EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Fluorescent immunohistochemistry of articular cartilage. Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA later /EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Immunohistochemistry, Staining

    In situ hybridization for Prg4 and Col2a1 . Cryosections from tibia decalcified with EDTA (A,B) or RNA later /EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35 S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1 . Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: In situ hybridization for Prg4 and Col2a1 . Cryosections from tibia decalcified with EDTA (A,B) or RNA later /EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35 S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1 . Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: In Situ Hybridization, Labeling, Expressing

    Cartilage morphology after EDTA or RNA later /EDTA decalcification. Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNA later /10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNA later /10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Cartilage morphology after EDTA or RNA later /EDTA decalcification. Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNA later /10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNA later /10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Staining

    Analysis of RNA integrity. Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA later /EDTA at pH 9.2 (C, D) or RNA later /EDTA at pH5.2 (E, F).

    Journal: PLoS ONE

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    doi: 10.1371/journal.pone.0058154

    Figure Lengend Snippet: Analysis of RNA integrity. Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA later /EDTA at pH 9.2 (C, D) or RNA later /EDTA at pH5.2 (E, F).

    Article Snippet: Quantitative PCR RNA isolated from EDTA or RNAlater /EDTA decalcified whole tibiae (500 ng total RNA) was reverse-transcribed as per the manufacturer’s protocol in a 20µL reaction using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Germany).

    Techniques: Electrophoresis, Isolation