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Millipore pbs t
(Top to Bottom) a: blot result of positive control B-Actin; b, c, d, and e are blot results of Core, E1, NS4A, NS4B respectively developed by AP conjugated Anti mouse with NBT/BCIP substrate (sigma) . Cells were lysed and protein was extracted after 72 hrs after transfection for single stable clone after 3 weeks About 80-100 μg of total protein were loaded into each well on 12.5% SDS-PAGE and electrophoretically blotted onto a Hybond-C extra nitrocellulose membrane semi-dry blotting apparatus. The membrane was blocked for 1 hour with a 5% milk solution in Phosphate Buffered Saline-0.05% Tween <t>(PBS-T),</t> washed three times with 50 ml of PBS-T. A mixture of primary antibodies for structural genes core (sc-57800), E1 (sc-65459) and non structural gene NS4A (sc-52415), NS4B (sc-65457) was added at a concentration of 1:500-1:800 in 5 ml of PBS-T. After incubating at room temperature for 1 hour, the membrane was washed 3 times with PBS-T. A secondary antibody, rabbit anti-mouse IgG, conjugated to alkaline phosphatase was added at a dilution of 1/1000 in PBS-T, incubated at room temperature for one hour. The membrane was washed for three times with PBS-T. Substrate tablet (NBT/BCIP) was dissolved in 1XPBS and blot was incubated for 15-30 min .
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1) Product Images from "Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs"

Article Title: Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs

Journal: Genetic Vaccines and Therapy

doi: 10.1186/1479-0556-9-12

(Top to Bottom) a: blot result of positive control B-Actin; b, c, d, and e are blot results of Core, E1, NS4A, NS4B respectively developed by AP conjugated Anti mouse with NBT/BCIP substrate (sigma) . Cells were lysed and protein was extracted after 72 hrs after transfection for single stable clone after 3 weeks About 80-100 μg of total protein were loaded into each well on 12.5% SDS-PAGE and electrophoretically blotted onto a Hybond-C extra nitrocellulose membrane semi-dry blotting apparatus. The membrane was blocked for 1 hour with a 5% milk solution in Phosphate Buffered Saline-0.05% Tween (PBS-T), washed three times with 50 ml of PBS-T. A mixture of primary antibodies for structural genes core (sc-57800), E1 (sc-65459) and non structural gene NS4A (sc-52415), NS4B (sc-65457) was added at a concentration of 1:500-1:800 in 5 ml of PBS-T. After incubating at room temperature for 1 hour, the membrane was washed 3 times with PBS-T. A secondary antibody, rabbit anti-mouse IgG, conjugated to alkaline phosphatase was added at a dilution of 1/1000 in PBS-T, incubated at room temperature for one hour. The membrane was washed for three times with PBS-T. Substrate tablet (NBT/BCIP) was dissolved in 1XPBS and blot was incubated for 15-30 min .
Figure Legend Snippet: (Top to Bottom) a: blot result of positive control B-Actin; b, c, d, and e are blot results of Core, E1, NS4A, NS4B respectively developed by AP conjugated Anti mouse with NBT/BCIP substrate (sigma) . Cells were lysed and protein was extracted after 72 hrs after transfection for single stable clone after 3 weeks About 80-100 μg of total protein were loaded into each well on 12.5% SDS-PAGE and electrophoretically blotted onto a Hybond-C extra nitrocellulose membrane semi-dry blotting apparatus. The membrane was blocked for 1 hour with a 5% milk solution in Phosphate Buffered Saline-0.05% Tween (PBS-T), washed three times with 50 ml of PBS-T. A mixture of primary antibodies for structural genes core (sc-57800), E1 (sc-65459) and non structural gene NS4A (sc-52415), NS4B (sc-65457) was added at a concentration of 1:500-1:800 in 5 ml of PBS-T. After incubating at room temperature for 1 hour, the membrane was washed 3 times with PBS-T. A secondary antibody, rabbit anti-mouse IgG, conjugated to alkaline phosphatase was added at a dilution of 1/1000 in PBS-T, incubated at room temperature for one hour. The membrane was washed for three times with PBS-T. Substrate tablet (NBT/BCIP) was dissolved in 1XPBS and blot was incubated for 15-30 min .

Techniques Used: Positive Control, Transfection, Stable Transfection, SDS Page, Semi Dry Blot, Concentration Assay, Incubation

2) Product Images from "Dephosphorylation at a Conserved SP Motif Governs cAMP Sensitivity and Nuclear Localization of Class IIa Histone Deacetylases *"

Article Title: Dephosphorylation at a Conserved SP Motif Governs cAMP Sensitivity and Nuclear Localization of Class IIa Histone Deacetylases *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.445668

Ser-266 is a novel phosphorylation site regulated by cAMP signaling. A , different amounts of regular peptide ( top ) or phosphopeptide ( bottom ) were spotted on a nitrocellulose membrane, air-dried, blocked for 2 h in PBS-T containing 20% horse serum, and
Figure Legend Snippet: Ser-266 is a novel phosphorylation site regulated by cAMP signaling. A , different amounts of regular peptide ( top ) or phosphopeptide ( bottom ) were spotted on a nitrocellulose membrane, air-dried, blocked for 2 h in PBS-T containing 20% horse serum, and

Techniques Used:

3) Product Images from "Epstein Barr Virus and Mycobacterium avium subsp. paratuberculosis peptides are recognized in sera and cerebrospinal fluid of MS patients"

Article Title: Epstein Barr Virus and Mycobacterium avium subsp. paratuberculosis peptides are recognized in sera and cerebrospinal fluid of MS patients

Journal: Scientific Reports

doi: 10.1038/srep22401

Competition assay with MBP ( A ) and IRF5 ( B ) coated ELISA plates. ( A ) CSF from 2 MS patients and 1 IND were pre-incubated overnight with saturating concentrations [10 μM] of MAP2694 38–46 (negative control), EBNA1 400–413 , MAP 106 121–132 and MBP 85–98 (positive control), The first bar represents a regularly performed ELISA (1:2 CSF in PBS-T) peptide. ( B ) The same CSF were pre-incubated with MAP2694 38–46 (negative control), BOLF1 305–320 , MAP_4027 18–32, and IRF5 424–434 (positive control). The first bar represents a regularly performed ELISA (1:2 CSF in PBS-T) peptide.
Figure Legend Snippet: Competition assay with MBP ( A ) and IRF5 ( B ) coated ELISA plates. ( A ) CSF from 2 MS patients and 1 IND were pre-incubated overnight with saturating concentrations [10 μM] of MAP2694 38–46 (negative control), EBNA1 400–413 , MAP 106 121–132 and MBP 85–98 (positive control), The first bar represents a regularly performed ELISA (1:2 CSF in PBS-T) peptide. ( B ) The same CSF were pre-incubated with MAP2694 38–46 (negative control), BOLF1 305–320 , MAP_4027 18–32, and IRF5 424–434 (positive control). The first bar represents a regularly performed ELISA (1:2 CSF in PBS-T) peptide.

Techniques Used: Competitive Binding Assay, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Incubation, Negative Control, Positive Control

4) Product Images from "A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B"

Article Title: A Single-Stranded DNA Aptamer That Selectively Binds to Staphylococcus aureus Enterotoxin B

Journal: PLoS ONE

doi: 10.1371/journal.pone.0033410

APT SEB1 binds to SEB, but not BSA. Aptamer-precipitation of SEB from 10-fold excess of BSA using several DNA sequences was visualized by 4–12% SDS-PAGE with silver stain. Dynabeads® M-270 Streptavidin magnetic beads coated with APT SEB1 (lane 3), random 78-base ssDNA (lanes 4–6), PCR forward primer (used in this study, lane 7), and nothing (lane 8), were incubated in 500 µl PBS-T incurred with 10 µg BSA and 1 µg SEB. After washing the Dynabeads with PBS-T, the protein eluate (lanes 3–8) was loaded onto the SDS-PAGE gel. Lanes 1 and 2 contain 50 ng of standard BSA and SEB, respectively. The protein bands labeled as “SA” represent the monomer of streptavidin liberated by the elution protocol.
Figure Legend Snippet: APT SEB1 binds to SEB, but not BSA. Aptamer-precipitation of SEB from 10-fold excess of BSA using several DNA sequences was visualized by 4–12% SDS-PAGE with silver stain. Dynabeads® M-270 Streptavidin magnetic beads coated with APT SEB1 (lane 3), random 78-base ssDNA (lanes 4–6), PCR forward primer (used in this study, lane 7), and nothing (lane 8), were incubated in 500 µl PBS-T incurred with 10 µg BSA and 1 µg SEB. After washing the Dynabeads with PBS-T, the protein eluate (lanes 3–8) was loaded onto the SDS-PAGE gel. Lanes 1 and 2 contain 50 ng of standard BSA and SEB, respectively. The protein bands labeled as “SA” represent the monomer of streptavidin liberated by the elution protocol.

Techniques Used: SDS Page, Silver Staining, Magnetic Beads, Polymerase Chain Reaction, Incubation, Labeling

APT SEB1 is selective for SEB within a complex mixture. The toxin-rich cell-free culture supernatant from four S. aureus strains was assayed for the presence of SEB by aptamer-precipitation. Five microliters of each culture supernatant was loaded onto an 4–12% SDS-PAGE gel to determine the protein content (lanes 2, 4, 6, 8). Three milliliters of each culture supernatant was incubated with APT SEB1 -coated Dynabeads. After washing with PBS-T, the resultant protein eluate from the APT SEB1 -coated Dynabeads was analyzed (lanes 3, 5, 7, 9). By PCR and ELISA analysis (Sandra Tallent, personal communication) the four strains potentially express a total of 17 enterotoxins and toxic shock syndrome toxin. However, only strain BAA1747 contains the gene for SEB. The protein bands labeled as “SA” represent the monomer of streptavidin liberated by the elution protocol.
Figure Legend Snippet: APT SEB1 is selective for SEB within a complex mixture. The toxin-rich cell-free culture supernatant from four S. aureus strains was assayed for the presence of SEB by aptamer-precipitation. Five microliters of each culture supernatant was loaded onto an 4–12% SDS-PAGE gel to determine the protein content (lanes 2, 4, 6, 8). Three milliliters of each culture supernatant was incubated with APT SEB1 -coated Dynabeads. After washing with PBS-T, the resultant protein eluate from the APT SEB1 -coated Dynabeads was analyzed (lanes 3, 5, 7, 9). By PCR and ELISA analysis (Sandra Tallent, personal communication) the four strains potentially express a total of 17 enterotoxins and toxic shock syndrome toxin. However, only strain BAA1747 contains the gene for SEB. The protein bands labeled as “SA” represent the monomer of streptavidin liberated by the elution protocol.

Techniques Used: SDS Page, Incubation, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Labeling

APT SEB1 is selective for SEB but not other closely related enterotoxins. Aptamer-precipitation of SEB from a mixture of enterotoxins was visualized by 4–12% SDS-PAGE with silver stain. Dynabeads® M-270 Streptavidin magnetic beads coated with APT SEB1 were incubated in 1000 µl PBS-T incurred with 1 µg each of SEA, SEC 1 , SEC 2 , SEC 3 , SED, and SEE. The aptamer-precipitation was carried out either with (lane 3) or without (lane 4) 1 µg SEB present in the mixture. After washing the Dynabeads with PBS-T, the protein eluate (lanes 3–8) was loaded onto the SDS-PAGE gel. Lanes 1 and 2 contain 200 ng of standard BSA and SEB, respectively. The protein bands labeled as “SA” represent the monomer of streptavidin liberated by the elution protocol.
Figure Legend Snippet: APT SEB1 is selective for SEB but not other closely related enterotoxins. Aptamer-precipitation of SEB from a mixture of enterotoxins was visualized by 4–12% SDS-PAGE with silver stain. Dynabeads® M-270 Streptavidin magnetic beads coated with APT SEB1 were incubated in 1000 µl PBS-T incurred with 1 µg each of SEA, SEC 1 , SEC 2 , SEC 3 , SED, and SEE. The aptamer-precipitation was carried out either with (lane 3) or without (lane 4) 1 µg SEB present in the mixture. After washing the Dynabeads with PBS-T, the protein eluate (lanes 3–8) was loaded onto the SDS-PAGE gel. Lanes 1 and 2 contain 200 ng of standard BSA and SEB, respectively. The protein bands labeled as “SA” represent the monomer of streptavidin liberated by the elution protocol.

Techniques Used: SDS Page, Silver Staining, Magnetic Beads, Incubation, Size-exclusion Chromatography, Labeling

5) Product Images from "Basidiomycete metabolites attenuate virulence properties of Candida albicans in vitro"

Article Title: Basidiomycete metabolites attenuate virulence properties of Candida albicans in vitro

Journal: Mycoses

doi: 10.1111/j.1439-0507.2008.01515.x

(a) Effect of Aureoquinone on Sap-release of CBS 5982. Drug addition was performed every 48 h. The percentage of methanol used for dissolving the drug was 5% for a Aureoquinone concentration of 50 μg ml −1 and 3% for 30 μg ml −1 . Pos. Co. denotes positive control of CBS 5982 in Sap-induction medium; Co. + NaCl denotes control + NaCl and served as negative control along with PBS-T. Extinction was determined at 405 nm with a reference wave length of 492 nm. Data shown represent mean ± SD of four experiments. **: highly significant ( P ≤ 0.01); *: significant ( P ≤ 0.05); statistical significance was determined using Student’s t -test analysis. (b) Effect of Laccaridione B on Sap-release of CBS 5982. The percentage of methanol used for dissolving the drug was 10% for a Laccaridione B concentration of 50 μg ml −1 , 6% for 30 μg ml −1 and 2% for 10 μg ml −1 . Further details are as described in (a). (c) Effect of Laccaridione B on Sap-release of CBS 5982. Drug addition was performed once only at the start of the experiment.
Figure Legend Snippet: (a) Effect of Aureoquinone on Sap-release of CBS 5982. Drug addition was performed every 48 h. The percentage of methanol used for dissolving the drug was 5% for a Aureoquinone concentration of 50 μg ml −1 and 3% for 30 μg ml −1 . Pos. Co. denotes positive control of CBS 5982 in Sap-induction medium; Co. + NaCl denotes control + NaCl and served as negative control along with PBS-T. Extinction was determined at 405 nm with a reference wave length of 492 nm. Data shown represent mean ± SD of four experiments. **: highly significant ( P ≤ 0.01); *: significant ( P ≤ 0.05); statistical significance was determined using Student’s t -test analysis. (b) Effect of Laccaridione B on Sap-release of CBS 5982. The percentage of methanol used for dissolving the drug was 10% for a Laccaridione B concentration of 50 μg ml −1 , 6% for 30 μg ml −1 and 2% for 10 μg ml −1 . Further details are as described in (a). (c) Effect of Laccaridione B on Sap-release of CBS 5982. Drug addition was performed once only at the start of the experiment.

Techniques Used: Concentration Assay, Positive Control, Negative Control

6) Product Images from "Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs"

Article Title: Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs

Journal: Genetic Vaccines and Therapy

doi: 10.1186/1479-0556-9-12

(Top to Bottom) a: blot result of positive control B-Actin; b, c, d, and e are blot results of Core, E1, NS4A, NS4B respectively developed by AP conjugated Anti mouse with NBT/BCIP substrate (sigma) . Cells were lysed and protein was extracted after 72 hrs after transfection for single stable clone after 3 weeks About 80-100 μg of total protein were loaded into each well on 12.5% SDS-PAGE and electrophoretically blotted onto a Hybond-C extra nitrocellulose membrane semi-dry blotting apparatus. The membrane was blocked for 1 hour with a 5% milk solution in Phosphate Buffered Saline-0.05% Tween (PBS-T), washed three times with 50 ml of PBS-T. A mixture of primary antibodies for structural genes core (sc-57800), E1 (sc-65459) and non structural gene NS4A (sc-52415), NS4B (sc-65457) was added at a concentration of 1:500-1:800 in 5 ml of PBS-T. After incubating at room temperature for 1 hour, the membrane was washed 3 times with PBS-T. A secondary antibody, rabbit anti-mouse IgG, conjugated to alkaline phosphatase was added at a dilution of 1/1000 in PBS-T, incubated at room temperature for one hour. The membrane was washed for three times with PBS-T. Substrate tablet (NBT/BCIP) was dissolved in 1XPBS and blot was incubated for 15-30 min .
Figure Legend Snippet: (Top to Bottom) a: blot result of positive control B-Actin; b, c, d, and e are blot results of Core, E1, NS4A, NS4B respectively developed by AP conjugated Anti mouse with NBT/BCIP substrate (sigma) . Cells were lysed and protein was extracted after 72 hrs after transfection for single stable clone after 3 weeks About 80-100 μg of total protein were loaded into each well on 12.5% SDS-PAGE and electrophoretically blotted onto a Hybond-C extra nitrocellulose membrane semi-dry blotting apparatus. The membrane was blocked for 1 hour with a 5% milk solution in Phosphate Buffered Saline-0.05% Tween (PBS-T), washed three times with 50 ml of PBS-T. A mixture of primary antibodies for structural genes core (sc-57800), E1 (sc-65459) and non structural gene NS4A (sc-52415), NS4B (sc-65457) was added at a concentration of 1:500-1:800 in 5 ml of PBS-T. After incubating at room temperature for 1 hour, the membrane was washed 3 times with PBS-T. A secondary antibody, rabbit anti-mouse IgG, conjugated to alkaline phosphatase was added at a dilution of 1/1000 in PBS-T, incubated at room temperature for one hour. The membrane was washed for three times with PBS-T. Substrate tablet (NBT/BCIP) was dissolved in 1XPBS and blot was incubated for 15-30 min .

Techniques Used: Positive Control, Transfection, Stable Transfection, SDS Page, Semi Dry Blot, Concentration Assay, Incubation

7) Product Images from "Curcumin inhibits the TGF-β1-dependent differentiation of lung fibroblasts via PPARγ-driven upregulation of cathepsins B and L"

Article Title: Curcumin inhibits the TGF-β1-dependent differentiation of lung fibroblasts via PPARγ-driven upregulation of cathepsins B and L

Journal: Scientific Reports

doi: 10.1038/s41598-018-36858-3

Expression level of cathepsins B and L in CCD-19Lu myofibroblasts treated by curcumin. Three days after induction of the differentiation of CCD-19l-Lu cells by TGF-β1 (5 ng/ml), curcumin (0–10 µM) was added for 48 h. ( a ) Quantitative real time PCR analysis of CatB and CatL. mRNA levels are normalized and expressed as percentage relative to untreated control (n = 3). ( b ) The related peptidase activity of secreted cysteine cathepsins was measured using Z-Phe-Arg-AMC (50 µM) as substrate. Results (corresponding to the release of fluorescent AMC, reported as arbitrary unit) are normalized and expressed as percentage relative to control in the absence of curcumin treatment (n = 3). Active site labeling of extracellular cathepsins by Biotinyl-(PEG) 2 -Ahx-LVG-DMK. Culture media of CCD-19Lu cells were incubated for 1 h with the activity-based probe (10 µM) at 30 °C according to 49 . Samples were subjected to electrophoresis on 12% SDS-PAGE under reducing conditions, electrotransferred to a nitrocellulose membrane, then blocked with 3% BSA in PBS-T. After incubation with an extravidin-peroxydase conjugate (1:3000, Sigma Aldrich) 2 h at room temperature, active cathepsins (lanes: 0, 2, 5, and 10 µM curcumin) were stained by chimiluminescence (ECL Plus Western Blotting Detection system). Full-length blots are presented in Supplementary Fig. 5 . ( c ) Two days after addition of curcumin, myofibroblasts layers were lysed, and the expression of intracellular CatB and CatL was analyzed by western blotting. A representative sample is shown (n = 3). White arrows indicate mature forms; black arrows correspond to pro-CatB and pro-CatL. β-actin was used for load control. Full-length blots are presented in Supplementary Fig. 5 . ( d ) Densitometric analysis of the protein level of intracellular mature CatB and CatL (normalized data relative to control without curcumin, n = 3). ( e ) Two days after treatment with curcumin, the protein level of extracellular CatB and CatL was analyzed by WB. A representative sample is shown (n = 3, white arrows, mature proteases). Full-length blots are presented in Supplementary Fig. 5 . ( f ) Corresponding densitometric analysis of extracellular mature CatB and CatL. Normalized data relative to control without curcumin (n = 3).
Figure Legend Snippet: Expression level of cathepsins B and L in CCD-19Lu myofibroblasts treated by curcumin. Three days after induction of the differentiation of CCD-19l-Lu cells by TGF-β1 (5 ng/ml), curcumin (0–10 µM) was added for 48 h. ( a ) Quantitative real time PCR analysis of CatB and CatL. mRNA levels are normalized and expressed as percentage relative to untreated control (n = 3). ( b ) The related peptidase activity of secreted cysteine cathepsins was measured using Z-Phe-Arg-AMC (50 µM) as substrate. Results (corresponding to the release of fluorescent AMC, reported as arbitrary unit) are normalized and expressed as percentage relative to control in the absence of curcumin treatment (n = 3). Active site labeling of extracellular cathepsins by Biotinyl-(PEG) 2 -Ahx-LVG-DMK. Culture media of CCD-19Lu cells were incubated for 1 h with the activity-based probe (10 µM) at 30 °C according to 49 . Samples were subjected to electrophoresis on 12% SDS-PAGE under reducing conditions, electrotransferred to a nitrocellulose membrane, then blocked with 3% BSA in PBS-T. After incubation with an extravidin-peroxydase conjugate (1:3000, Sigma Aldrich) 2 h at room temperature, active cathepsins (lanes: 0, 2, 5, and 10 µM curcumin) were stained by chimiluminescence (ECL Plus Western Blotting Detection system). Full-length blots are presented in Supplementary Fig. 5 . ( c ) Two days after addition of curcumin, myofibroblasts layers were lysed, and the expression of intracellular CatB and CatL was analyzed by western blotting. A representative sample is shown (n = 3). White arrows indicate mature forms; black arrows correspond to pro-CatB and pro-CatL. β-actin was used for load control. Full-length blots are presented in Supplementary Fig. 5 . ( d ) Densitometric analysis of the protein level of intracellular mature CatB and CatL (normalized data relative to control without curcumin, n = 3). ( e ) Two days after treatment with curcumin, the protein level of extracellular CatB and CatL was analyzed by WB. A representative sample is shown (n = 3, white arrows, mature proteases). Full-length blots are presented in Supplementary Fig. 5 . ( f ) Corresponding densitometric analysis of extracellular mature CatB and CatL. Normalized data relative to control without curcumin (n = 3).

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Labeling, Incubation, Electrophoresis, SDS Page, Staining, Western Blot

8) Product Images from "A Monoclonal Antibody-Based Copro-ELISA Kit for Canine Echinococcosis to Support the PAHO Effort for Hydatid Disease Control in South America"

Article Title: A Monoclonal Antibody-Based Copro-ELISA Kit for Canine Echinococcosis to Support the PAHO Effort for Hydatid Disease Control in South America

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0001967

Time-course stability of the copro-ELISA kit components. A) Dried or freshly-coated plates (white and gray symbols, respectively) were tested at different time points by assaying a set of 3 weak-positive and 3 negative samples (average = circles and squares, respectively). B–D) Fresh solutions of the calibrator were tested in triplicate at different time points, using fresh (grey circles) or stored dilutions of the kit reagents kept at room temperature (white) or 37°C (black symbols). All values were normalized with regard to the value obtained with the fresh reagent. B) Stability of the calibrator: triangles, PBS, 5% glycerol, 0.1% Kathon. C) MAb Eg9 stability: squares, PBS-T; triangles, PBS-T, 0.1% Kathon. D) Stability of the peroxidase anti-mouse IgG antibody: squares, PBS, 0.1% BSA; triangles, PBS, 0.1% BSA, 0.1% Kathon, 0.1 mM 3,3′,5,5′-tetramethylbenzidine (TMB).
Figure Legend Snippet: Time-course stability of the copro-ELISA kit components. A) Dried or freshly-coated plates (white and gray symbols, respectively) were tested at different time points by assaying a set of 3 weak-positive and 3 negative samples (average = circles and squares, respectively). B–D) Fresh solutions of the calibrator were tested in triplicate at different time points, using fresh (grey circles) or stored dilutions of the kit reagents kept at room temperature (white) or 37°C (black symbols). All values were normalized with regard to the value obtained with the fresh reagent. B) Stability of the calibrator: triangles, PBS, 5% glycerol, 0.1% Kathon. C) MAb Eg9 stability: squares, PBS-T; triangles, PBS-T, 0.1% Kathon. D) Stability of the peroxidase anti-mouse IgG antibody: squares, PBS, 0.1% BSA; triangles, PBS, 0.1% BSA, 0.1% Kathon, 0.1 mM 3,3′,5,5′-tetramethylbenzidine (TMB).

Techniques Used: Enzyme-linked Immunosorbent Assay

9) Product Images from "Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs"

Article Title: Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs

Journal: Genetic Vaccines and Therapy

doi: 10.1186/1479-0556-9-12

(Top to Bottom) a: blot result of positive control B-Actin; b, c, d, and e are blot results of Core, E1, NS4A, NS4B respectively developed by AP conjugated Anti mouse with NBT/BCIP substrate (sigma) . Cells were lysed and protein was extracted after 72 hrs after transfection for single stable clone after 3 weeks About 80-100 μg of total protein were loaded into each well on 12.5% SDS-PAGE and electrophoretically blotted onto a Hybond-C extra nitrocellulose membrane semi-dry blotting apparatus. The membrane was blocked for 1 hour with a 5% milk solution in Phosphate Buffered Saline-0.05% Tween (PBS-T), washed three times with 50 ml of PBS-T. A mixture of primary antibodies for structural genes core (sc-57800), E1 (sc-65459) and non structural gene NS4A (sc-52415), NS4B (sc-65457) was added at a concentration of 1:500-1:800 in 5 ml of PBS-T. After incubating at room temperature for 1 hour, the membrane was washed 3 times with PBS-T. A secondary antibody, rabbit anti-mouse IgG, conjugated to alkaline phosphatase was added at a dilution of 1/1000 in PBS-T, incubated at room temperature for one hour. The membrane was washed for three times with PBS-T. Substrate tablet (NBT/BCIP) was dissolved in 1XPBS and blot was incubated for 15-30 min .
Figure Legend Snippet: (Top to Bottom) a: blot result of positive control B-Actin; b, c, d, and e are blot results of Core, E1, NS4A, NS4B respectively developed by AP conjugated Anti mouse with NBT/BCIP substrate (sigma) . Cells were lysed and protein was extracted after 72 hrs after transfection for single stable clone after 3 weeks About 80-100 μg of total protein were loaded into each well on 12.5% SDS-PAGE and electrophoretically blotted onto a Hybond-C extra nitrocellulose membrane semi-dry blotting apparatus. The membrane was blocked for 1 hour with a 5% milk solution in Phosphate Buffered Saline-0.05% Tween (PBS-T), washed three times with 50 ml of PBS-T. A mixture of primary antibodies for structural genes core (sc-57800), E1 (sc-65459) and non structural gene NS4A (sc-52415), NS4B (sc-65457) was added at a concentration of 1:500-1:800 in 5 ml of PBS-T. After incubating at room temperature for 1 hour, the membrane was washed 3 times with PBS-T. A secondary antibody, rabbit anti-mouse IgG, conjugated to alkaline phosphatase was added at a dilution of 1/1000 in PBS-T, incubated at room temperature for one hour. The membrane was washed for three times with PBS-T. Substrate tablet (NBT/BCIP) was dissolved in 1XPBS and blot was incubated for 15-30 min .

Techniques Used: Positive Control, Transfection, Stable Transfection, SDS Page, Semi Dry Blot, Concentration Assay, Incubation

10) Product Images from "Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs"

Article Title: Establishment of stable Huh-7 cell lines expressing various hepatitis C virus genotype 3a protein: an in-vitro testing system for novel anti-HCV drugs

Journal: Genetic Vaccines and Therapy

doi: 10.1186/1479-0556-9-12

(Top to Bottom) a: blot result of positive control B-Actin; b, c, d, and e are blot results of Core, E1, NS4A, NS4B respectively developed by AP conjugated Anti mouse with NBT/BCIP substrate (sigma) . Cells were lysed and protein was extracted after 72 hrs after transfection for single stable clone after 3 weeks About 80-100 μg of total protein were loaded into each well on 12.5% SDS-PAGE and electrophoretically blotted onto a Hybond-C extra nitrocellulose membrane semi-dry blotting apparatus. The membrane was blocked for 1 hour with a 5% milk solution in Phosphate Buffered Saline-0.05% Tween (PBS-T), washed three times with 50 ml of PBS-T. A mixture of primary antibodies for structural genes core (sc-57800), E1 (sc-65459) and non structural gene NS4A (sc-52415), NS4B (sc-65457) was added at a concentration of 1:500-1:800 in 5 ml of PBS-T. After incubating at room temperature for 1 hour, the membrane was washed 3 times with PBS-T. A secondary antibody, rabbit anti-mouse IgG, conjugated to alkaline phosphatase was added at a dilution of 1/1000 in PBS-T, incubated at room temperature for one hour. The membrane was washed for three times with PBS-T. Substrate tablet (NBT/BCIP) was dissolved in 1XPBS and blot was incubated for 15-30 min .
Figure Legend Snippet: (Top to Bottom) a: blot result of positive control B-Actin; b, c, d, and e are blot results of Core, E1, NS4A, NS4B respectively developed by AP conjugated Anti mouse with NBT/BCIP substrate (sigma) . Cells were lysed and protein was extracted after 72 hrs after transfection for single stable clone after 3 weeks About 80-100 μg of total protein were loaded into each well on 12.5% SDS-PAGE and electrophoretically blotted onto a Hybond-C extra nitrocellulose membrane semi-dry blotting apparatus. The membrane was blocked for 1 hour with a 5% milk solution in Phosphate Buffered Saline-0.05% Tween (PBS-T), washed three times with 50 ml of PBS-T. A mixture of primary antibodies for structural genes core (sc-57800), E1 (sc-65459) and non structural gene NS4A (sc-52415), NS4B (sc-65457) was added at a concentration of 1:500-1:800 in 5 ml of PBS-T. After incubating at room temperature for 1 hour, the membrane was washed 3 times with PBS-T. A secondary antibody, rabbit anti-mouse IgG, conjugated to alkaline phosphatase was added at a dilution of 1/1000 in PBS-T, incubated at room temperature for one hour. The membrane was washed for three times with PBS-T. Substrate tablet (NBT/BCIP) was dissolved in 1XPBS and blot was incubated for 15-30 min .

Techniques Used: Positive Control, Transfection, Stable Transfection, SDS Page, Semi Dry Blot, Concentration Assay, Incubation

11) Product Images from "Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis"

Article Title: Antigenicity, Immunogenicity and Protective Efficacy of Three Proteins Expressed in the Promastigote and Amastigote Stages of Leishmania infantum against Visceral Leishmaniasis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0137683

Immunoblotting experiments using the recombinant proteins and a canine serological panel. For immunoblotting experiments, the rLiHyp1 (36.6 kDa), rLiHyp6 (21.4 kDa), and rHRF (19.4 kDa) proteins (10 μg, each) were submitted to a 12% SDS-PAGE and blotted onto a nitrocellulose membrane, which were blocked with a PBS-T containing 5% BSA solution, and incubated with a pool of sera of asymptomatic and symptomatic VL dogs, or with a pool containing sera of non-infected dogs (1:200 and 1:100 diluted in PBS-T, respectively). Peroxidase conjugated anti-dog IgG (1:10,000) was used as a second antibody. The reactivities against the rLiHyp1, rLiHyp6 and rHRF proteins are shown. A low range protein ladder (Invitrogen TM , Life Technologies, USA) was used (Mr). The individual reactions of the rLiHyp1, rLiHyp6, and rHRF proteins with the pools of sera from non-infected or L . infantum -infected dogs (NI and CVL, respectively) are shown. Immunoblottings were derived from three independent experiments, and one representative preparation is showed in this study.
Figure Legend Snippet: Immunoblotting experiments using the recombinant proteins and a canine serological panel. For immunoblotting experiments, the rLiHyp1 (36.6 kDa), rLiHyp6 (21.4 kDa), and rHRF (19.4 kDa) proteins (10 μg, each) were submitted to a 12% SDS-PAGE and blotted onto a nitrocellulose membrane, which were blocked with a PBS-T containing 5% BSA solution, and incubated with a pool of sera of asymptomatic and symptomatic VL dogs, or with a pool containing sera of non-infected dogs (1:200 and 1:100 diluted in PBS-T, respectively). Peroxidase conjugated anti-dog IgG (1:10,000) was used as a second antibody. The reactivities against the rLiHyp1, rLiHyp6 and rHRF proteins are shown. A low range protein ladder (Invitrogen TM , Life Technologies, USA) was used (Mr). The individual reactions of the rLiHyp1, rLiHyp6, and rHRF proteins with the pools of sera from non-infected or L . infantum -infected dogs (NI and CVL, respectively) are shown. Immunoblottings were derived from three independent experiments, and one representative preparation is showed in this study.

Techniques Used: Recombinant, SDS Page, Incubation, Infection, Derivative Assay

12) Product Images from "Epstein Barr Virus and Mycobacterium avium subsp. paratuberculosis peptides are recognized in sera and cerebrospinal fluid of MS patients"

Article Title: Epstein Barr Virus and Mycobacterium avium subsp. paratuberculosis peptides are recognized in sera and cerebrospinal fluid of MS patients

Journal: Scientific Reports

doi: 10.1038/srep22401

Competition assay with MBP ( A ) and IRF5 ( B ) coated ELISA plates. ( A ) CSF from 2 MS patients and 1 IND were pre-incubated overnight with saturating concentrations [10 μM] of MAP2694 38–46 (negative control), EBNA1 400–413 , MAP 106 121–132 and MBP 85–98 (positive control), The first bar represents a regularly performed ELISA (1:2 CSF in PBS-T) peptide. ( B ) The same CSF were pre-incubated with MAP2694 38–46 (negative control), BOLF1 305–320 , MAP_4027 18–32, and IRF5 424–434 (positive control). The first bar represents a regularly performed ELISA (1:2 CSF in PBS-T) peptide.
Figure Legend Snippet: Competition assay with MBP ( A ) and IRF5 ( B ) coated ELISA plates. ( A ) CSF from 2 MS patients and 1 IND were pre-incubated overnight with saturating concentrations [10 μM] of MAP2694 38–46 (negative control), EBNA1 400–413 , MAP 106 121–132 and MBP 85–98 (positive control), The first bar represents a regularly performed ELISA (1:2 CSF in PBS-T) peptide. ( B ) The same CSF were pre-incubated with MAP2694 38–46 (negative control), BOLF1 305–320 , MAP_4027 18–32, and IRF5 424–434 (positive control). The first bar represents a regularly performed ELISA (1:2 CSF in PBS-T) peptide.

Techniques Used: Competitive Binding Assay, Enzyme-linked Immunosorbent Assay, Mass Spectrometry, Incubation, Negative Control, Positive Control

13) Product Images from "Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics"

Article Title: Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics

Journal: Sensors and Actuators. B, Chemical

doi: 10.1016/j.snb.2005.07.033

Exemplary fluorescence image and data interpretation of antibody detection with the peptide microarray. (a) PNACs of the six peptide probes, spotting control (Dy-633 labeled NA) and NA reference spots were spotted in 0.4 mg ml −1 concentration, the incubation control (mouse IgG) in 0.2 mg ml −1 concentration in 10 mM PBS pH 7.6 onto amine coated glass slides. The slides were incubated 3 h at room temperature (PBS) or over night at +4 °C (1:50 diluted human serum) with a mixture of mAbs (1 μg ml −1 each) and 2 h with Cy5-GAM (10 μg ml −1 ) in PBS-T at room temperature. Fluorescence images were taken with the Affymetrix 428 ArrayScanner at 25 dB PMT adjustment. The scheme in the right panel displays the chip layout. (b) Quantitative fluorescence data interpretation. Signal/background ratios represent mean values of 32 spots in 8 arrays. The limit of detection was 2.7 for Myc-mAb, 2.5 for Pol-mAb and below 1.5 for other mAbs.
Figure Legend Snippet: Exemplary fluorescence image and data interpretation of antibody detection with the peptide microarray. (a) PNACs of the six peptide probes, spotting control (Dy-633 labeled NA) and NA reference spots were spotted in 0.4 mg ml −1 concentration, the incubation control (mouse IgG) in 0.2 mg ml −1 concentration in 10 mM PBS pH 7.6 onto amine coated glass slides. The slides were incubated 3 h at room temperature (PBS) or over night at +4 °C (1:50 diluted human serum) with a mixture of mAbs (1 μg ml −1 each) and 2 h with Cy5-GAM (10 μg ml −1 ) in PBS-T at room temperature. Fluorescence images were taken with the Affymetrix 428 ArrayScanner at 25 dB PMT adjustment. The scheme in the right panel displays the chip layout. (b) Quantitative fluorescence data interpretation. Signal/background ratios represent mean values of 32 spots in 8 arrays. The limit of detection was 2.7 for Myc-mAb, 2.5 for Pol-mAb and below 1.5 for other mAbs.

Techniques Used: Fluorescence, Peptide Microarray, Labeling, Concentration Assay, Incubation, Chromatin Immunoprecipitation

Evaluation of chip surface, spotting buffer and coupling kinetic. (a) Fluorescence images of Dy-633 NA immobilized on amine, aldehyde and epoxy coated glass slides in grey scale. NA was spotted in the specified concentrations (mg ml −1 ) in 10 mM PBS pH 7.6 and incubated for 24 h. Unbound NA was removed by washing for 1 h in PBS-T + 10% skim milk. Fluorescence images were taken with the Affymetrix 428 ArrayScanner at 5 dB voltage gain. (b) Quantitative spot analysis. Relative fluorescence units (RFU) represent the mean of 120 Spots in 12 arrays per slide type and NA concentration. (c) Time course of adsorptive surface coupling of NA on amine coated glass slides. NA was spotted in 0.4 mg ml −1 concentration in the specified buffers. Fluorescence intensities of spots (mean of 80 spots in 8 arrays per buffer and time) were related to protein density via a calibration curve.
Figure Legend Snippet: Evaluation of chip surface, spotting buffer and coupling kinetic. (a) Fluorescence images of Dy-633 NA immobilized on amine, aldehyde and epoxy coated glass slides in grey scale. NA was spotted in the specified concentrations (mg ml −1 ) in 10 mM PBS pH 7.6 and incubated for 24 h. Unbound NA was removed by washing for 1 h in PBS-T + 10% skim milk. Fluorescence images were taken with the Affymetrix 428 ArrayScanner at 5 dB voltage gain. (b) Quantitative spot analysis. Relative fluorescence units (RFU) represent the mean of 120 Spots in 12 arrays per slide type and NA concentration. (c) Time course of adsorptive surface coupling of NA on amine coated glass slides. NA was spotted in 0.4 mg ml −1 concentration in the specified buffers. Fluorescence intensities of spots (mean of 80 spots in 8 arrays per buffer and time) were related to protein density via a calibration curve.

Techniques Used: Chromatin Immunoprecipitation, Fluorescence, Incubation, Concentration Assay

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Co-Immunoprecipitation Assay:

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Sonication:

Article Title: Cloning, expression, purification and crystallization as well as X-ray fluorescence and preliminary X-ray diffraction analyses of human ADP-ribosylhydrolase 1
Article Snippet: .. The cells were suspended in 6 ml lysis buffer [50 m M NaH2 PO4 pH 8.0, 500 m M NaCl, 10 m M MgCl2 , 3 m M β-mercaptoethanol (β-ME), 1 m M 4-(2-aminoethyl)benzenesulfonylfluoride, 0.1%( v / v ) Triton X-­100, 0.1 µg ml−1 lysozyme and 12.5 U ml−1 Benzonase nuclease (Novagen)] per gram wet weight and incubated at 294 K for 30 min. Lysis was performed by sonication for 5 min in 15 s pulses at 277 K. Cell debris was removed by centrifugation at 15 500 g for 20 min at 277 K. 1 g Protino Ni–IDA matrix (Macherey-Nagel) was added per 50 ml crude lysate and the slurry was incubated for 1–2 h at 277 K in order to allow complete binding. ..

Protease Inhibitor:

Article Title: A Positive Role of c-Myc in Regulating Androgen Receptor and its Splice Variants in Prostate Cancer
Article Snippet: .. Co-immunoprecipitation assay Cells were pelleted, washed twice with cold PBS, and lysed in 500 μl of lysis buffer (1% NP-40, 20 mmol/L Tris, pH 7.4, 140 mmol/L NaCl, and 2 mmol/L EDTA) containing a protease inhibitor cocktail (Sigma). .. The lysate was then used for immunoprecipitation with a c-Myc antibody (Sigma) or an IgG control.

Article Title: Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE 1
Article Snippet: .. For SbSnRK1 phosphorylation, frozen samples were homogenized and incubated in lysis buffer (137 m m NaCl, 2.7 m m KCl, 10 m m Na2 HPO4 , 2 m m KH2 PO4 , pH 7.5, 1 m m EDTA, 0.05% [v/v] Triton X-100, 1:500 protease inhibitor cocktail [Sigma; P9599], and 1% [v/v] phosphatase inhibitor cocktail 3 [Sigma; P0044]) for 5 min. After centrifugation at 18,000 g at 4°C for 15 min, the protein concentration was quantified via the manufacturer’s protocol. .. Equal amounts of lysate (300 μg) were separated on a 10% (w/v) SDS-PAGE gel and then blotted onto a polyvinylidene difluoride membrane (0.45 μm; Millipore) that was analyzed with the phospho-AMPKα (Thr-172) polyclonal antibody (Cell Signaling Technology), which recognizes the highly conserved sequence of phosphorylated SbSnRK1 or PKIN1.

Article Title: Astroglial IFITM3 mediates neuronal impairments following neonatal immune challenge in mice
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Article Title: Maternal insulin resistance multigenerationally impairs synaptic plasticity and memory via gametic mechanisms
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Centrifugation:

Article Title: Cloning, expression, purification and crystallization as well as X-ray fluorescence and preliminary X-ray diffraction analyses of human ADP-ribosylhydrolase 1
Article Snippet: .. The cells were suspended in 6 ml lysis buffer [50 m M NaH2 PO4 pH 8.0, 500 m M NaCl, 10 m M MgCl2 , 3 m M β-mercaptoethanol (β-ME), 1 m M 4-(2-aminoethyl)benzenesulfonylfluoride, 0.1%( v / v ) Triton X-­100, 0.1 µg ml−1 lysozyme and 12.5 U ml−1 Benzonase nuclease (Novagen)] per gram wet weight and incubated at 294 K for 30 min. Lysis was performed by sonication for 5 min in 15 s pulses at 277 K. Cell debris was removed by centrifugation at 15 500 g for 20 min at 277 K. 1 g Protino Ni–IDA matrix (Macherey-Nagel) was added per 50 ml crude lysate and the slurry was incubated for 1–2 h at 277 K in order to allow complete binding. ..

Article Title: Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE 1
Article Snippet: .. For SbSnRK1 phosphorylation, frozen samples were homogenized and incubated in lysis buffer (137 m m NaCl, 2.7 m m KCl, 10 m m Na2 HPO4 , 2 m m KH2 PO4 , pH 7.5, 1 m m EDTA, 0.05% [v/v] Triton X-100, 1:500 protease inhibitor cocktail [Sigma; P9599], and 1% [v/v] phosphatase inhibitor cocktail 3 [Sigma; P0044]) for 5 min. After centrifugation at 18,000 g at 4°C for 15 min, the protein concentration was quantified via the manufacturer’s protocol. .. Equal amounts of lysate (300 μg) were separated on a 10% (w/v) SDS-PAGE gel and then blotted onto a polyvinylidene difluoride membrane (0.45 μm; Millipore) that was analyzed with the phospho-AMPKα (Thr-172) polyclonal antibody (Cell Signaling Technology), which recognizes the highly conserved sequence of phosphorylated SbSnRK1 or PKIN1.

Incubation:

Article Title: Cloning, expression, purification and crystallization as well as X-ray fluorescence and preliminary X-ray diffraction analyses of human ADP-ribosylhydrolase 1
Article Snippet: .. The cells were suspended in 6 ml lysis buffer [50 m M NaH2 PO4 pH 8.0, 500 m M NaCl, 10 m M MgCl2 , 3 m M β-mercaptoethanol (β-ME), 1 m M 4-(2-aminoethyl)benzenesulfonylfluoride, 0.1%( v / v ) Triton X-­100, 0.1 µg ml−1 lysozyme and 12.5 U ml−1 Benzonase nuclease (Novagen)] per gram wet weight and incubated at 294 K for 30 min. Lysis was performed by sonication for 5 min in 15 s pulses at 277 K. Cell debris was removed by centrifugation at 15 500 g for 20 min at 277 K. 1 g Protino Ni–IDA matrix (Macherey-Nagel) was added per 50 ml crude lysate and the slurry was incubated for 1–2 h at 277 K in order to allow complete binding. ..

Article Title: Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE 1
Article Snippet: .. For SbSnRK1 phosphorylation, frozen samples were homogenized and incubated in lysis buffer (137 m m NaCl, 2.7 m m KCl, 10 m m Na2 HPO4 , 2 m m KH2 PO4 , pH 7.5, 1 m m EDTA, 0.05% [v/v] Triton X-100, 1:500 protease inhibitor cocktail [Sigma; P9599], and 1% [v/v] phosphatase inhibitor cocktail 3 [Sigma; P0044]) for 5 min. After centrifugation at 18,000 g at 4°C for 15 min, the protein concentration was quantified via the manufacturer’s protocol. .. Equal amounts of lysate (300 μg) were separated on a 10% (w/v) SDS-PAGE gel and then blotted onto a polyvinylidene difluoride membrane (0.45 μm; Millipore) that was analyzed with the phospho-AMPKα (Thr-172) polyclonal antibody (Cell Signaling Technology), which recognizes the highly conserved sequence of phosphorylated SbSnRK1 or PKIN1.

Mouse Assay:

Article Title: Astroglial IFITM3 mediates neuronal impairments following neonatal immune challenge in mice
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Protein Extraction:

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Protein Concentration:

Article Title: Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE 1
Article Snippet: .. For SbSnRK1 phosphorylation, frozen samples were homogenized and incubated in lysis buffer (137 m m NaCl, 2.7 m m KCl, 10 m m Na2 HPO4 , 2 m m KH2 PO4 , pH 7.5, 1 m m EDTA, 0.05% [v/v] Triton X-100, 1:500 protease inhibitor cocktail [Sigma; P9599], and 1% [v/v] phosphatase inhibitor cocktail 3 [Sigma; P0044]) for 5 min. After centrifugation at 18,000 g at 4°C for 15 min, the protein concentration was quantified via the manufacturer’s protocol. .. Equal amounts of lysate (300 μg) were separated on a 10% (w/v) SDS-PAGE gel and then blotted onto a polyvinylidene difluoride membrane (0.45 μm; Millipore) that was analyzed with the phospho-AMPKα (Thr-172) polyclonal antibody (Cell Signaling Technology), which recognizes the highly conserved sequence of phosphorylated SbSnRK1 or PKIN1.

Western Blot:

Article Title: Maternal insulin resistance multigenerationally impairs synaptic plasticity and memory via gametic mechanisms
Article Snippet: .. Western blotting Tissues (hippocampi or ovaries) were lysed in ice-cold lysis buffer (NaCl 150 mM, Tris-HCl 50 mM pH 7.4, EDTA 2 mM) containing 1% Triton X-100, 0.1% SDS, 1 × protease inhibitor cocktail (Sigma-Aldrich), 1 mM sodium orthovanadate (Sigma-Aldrich), and 1 mM sodium fluoride (Sigma-Aldrich). .. Cells were incubated for 10 min on ice with occasional vortexing and spun down at 22,000 × g , 4 °C.

Lysis:

Article Title: A Positive Role of c-Myc in Regulating Androgen Receptor and its Splice Variants in Prostate Cancer
Article Snippet: .. Co-immunoprecipitation assay Cells were pelleted, washed twice with cold PBS, and lysed in 500 μl of lysis buffer (1% NP-40, 20 mmol/L Tris, pH 7.4, 140 mmol/L NaCl, and 2 mmol/L EDTA) containing a protease inhibitor cocktail (Sigma). .. The lysate was then used for immunoprecipitation with a c-Myc antibody (Sigma) or an IgG control.

Article Title: Cloning, expression, purification and crystallization as well as X-ray fluorescence and preliminary X-ray diffraction analyses of human ADP-ribosylhydrolase 1
Article Snippet: .. The cells were suspended in 6 ml lysis buffer [50 m M NaH2 PO4 pH 8.0, 500 m M NaCl, 10 m M MgCl2 , 3 m M β-mercaptoethanol (β-ME), 1 m M 4-(2-aminoethyl)benzenesulfonylfluoride, 0.1%( v / v ) Triton X-­100, 0.1 µg ml−1 lysozyme and 12.5 U ml−1 Benzonase nuclease (Novagen)] per gram wet weight and incubated at 294 K for 30 min. Lysis was performed by sonication for 5 min in 15 s pulses at 277 K. Cell debris was removed by centrifugation at 15 500 g for 20 min at 277 K. 1 g Protino Ni–IDA matrix (Macherey-Nagel) was added per 50 ml crude lysate and the slurry was incubated for 1–2 h at 277 K in order to allow complete binding. ..

Article Title: Subtle Regulation of Potato Acid Invertase Activity by a Protein Complex of Invertase, Invertase Inhibitor, and SUCROSE NONFERMENTING1-RELATED PROTEIN KINASE 1
Article Snippet: .. For SbSnRK1 phosphorylation, frozen samples were homogenized and incubated in lysis buffer (137 m m NaCl, 2.7 m m KCl, 10 m m Na2 HPO4 , 2 m m KH2 PO4 , pH 7.5, 1 m m EDTA, 0.05% [v/v] Triton X-100, 1:500 protease inhibitor cocktail [Sigma; P9599], and 1% [v/v] phosphatase inhibitor cocktail 3 [Sigma; P0044]) for 5 min. After centrifugation at 18,000 g at 4°C for 15 min, the protein concentration was quantified via the manufacturer’s protocol. .. Equal amounts of lysate (300 μg) were separated on a 10% (w/v) SDS-PAGE gel and then blotted onto a polyvinylidene difluoride membrane (0.45 μm; Millipore) that was analyzed with the phospho-AMPKα (Thr-172) polyclonal antibody (Cell Signaling Technology), which recognizes the highly conserved sequence of phosphorylated SbSnRK1 or PKIN1.

Article Title: Astroglial IFITM3 mediates neuronal impairments following neonatal immune challenge in mice
Article Snippet: .. The brains were collected from neonatal mice at 24 h after the final polyI:C treatment on PD7 and homogenized with lysis buffer (40 mM Tris‐HCl [pH 7.4], 2 mM EDTA, 100 mM NaCl, and 0.5% Triton X‐100, and protease inhibitor cocktail [Sigma‐Aldrich]). .. Lysate protein (1000 mg) collected from COS7 cells and neonatal mouse brain was added to Protein A/G Plus Agarose cross‐linked to rabbit anti‐HA (Sigma‐Aldrich), rabbit anti‐ATP6V0B (Abgent, San Diego, CA), mouse anti‐kinesin heavy chain (Millipore), rabbit anti‐visfatin (Bethyl Laboratories, Montgomery, TX), normal rabbit IgG, or normal mouse IgG (Santa Cruz Biotechnology), and incubated overnight at 4°C.

Article Title: Melatonin receptor signaling contributes to neuroprotection upon arousal from torpor in thirteen-lined ground squirrels
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Article Title: Maternal insulin resistance multigenerationally impairs synaptic plasticity and memory via gametic mechanisms
Article Snippet: .. Western blotting Tissues (hippocampi or ovaries) were lysed in ice-cold lysis buffer (NaCl 150 mM, Tris-HCl 50 mM pH 7.4, EDTA 2 mM) containing 1% Triton X-100, 0.1% SDS, 1 × protease inhibitor cocktail (Sigma-Aldrich), 1 mM sodium orthovanadate (Sigma-Aldrich), and 1 mM sodium fluoride (Sigma-Aldrich). .. Cells were incubated for 10 min on ice with occasional vortexing and spun down at 22,000 × g , 4 °C.

Binding Assay:

Article Title: Cloning, expression, purification and crystallization as well as X-ray fluorescence and preliminary X-ray diffraction analyses of human ADP-ribosylhydrolase 1
Article Snippet: .. The cells were suspended in 6 ml lysis buffer [50 m M NaH2 PO4 pH 8.0, 500 m M NaCl, 10 m M MgCl2 , 3 m M β-mercaptoethanol (β-ME), 1 m M 4-(2-aminoethyl)benzenesulfonylfluoride, 0.1%( v / v ) Triton X-­100, 0.1 µg ml−1 lysozyme and 12.5 U ml−1 Benzonase nuclease (Novagen)] per gram wet weight and incubated at 294 K for 30 min. Lysis was performed by sonication for 5 min in 15 s pulses at 277 K. Cell debris was removed by centrifugation at 15 500 g for 20 min at 277 K. 1 g Protino Ni–IDA matrix (Macherey-Nagel) was added per 50 ml crude lysate and the slurry was incubated for 1–2 h at 277 K in order to allow complete binding. ..

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