Whole cell protein extracts (protein lysates) were prepared using RIPA lysis buffer (Santa Cruz Biotechnology, sc-24948). Protein estimation was performed using the BCA kit. Equal amounts of protein from the total cell lysates (22 to 50 µg/lane) were separated by SDS-PAGE (4–20% gradient gel), and transferred to PVDF membrane using wet transfer. Depending upon the primary antibody to be used, the membranes were blocked in 5% skim milk (Cell Signaling Technology, 9999) or 5% bovine serum albumin (Cell Signaling Technology, 9998) for 1 h. The membranes were then probed overnight at 4 °C with specific primary antibodies diluted in 1% bovine serum albumin or 1% skim milk. Then, the membranes were washed and probed with the respective secondary isotype-specific antibodies tagged with horseradish peroxidase. Bound immuno-complexes were detected using WEST PICO chemiluminescence substrate and exposed to Hyperfilm-ECL. The same blots were reprobed with GAPDH or tubulin, α antibody as a loading control. Densitometry analysis for determining the band intensity was performed using ImageJ software.
Bovine serum albumin (bsa)
Manufactured by Cell Signaling Technology
Sourced in United States
Bovine serum albumin (BSA) is a protein derived from bovine blood serum. It is commonly used as a stabilizing agent, blocking agent, and protein standard in various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (16 mentions)
Triton x 100, by Merck Group (5 mentions)
Gapdh, by Cell Signaling Technology (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Nitrocellulose membrane, by GE Healthcare (2 mentions)
Gprc5a antibody, by Merck Group (2 mentions)
Ge imagequant las 4000, by GE Healthcare (2 mentions)
Pierce bca assay, by Thermo Fisher Scientific (2 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Fluoview 500 laser scanning confocal microscope, by Olympus (2 mentions)
Evans blue dye, by Merck Group (2 mentions)
Fluoview software, by Olympus (2 mentions)
Laser scanning confocal immunofluorescence microscope, by Zeiss (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
β actin antibody, by Cell Signaling Technology (2 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (2 mentions)
84 protocols using bovine serum albumin (bsa)
1
Western Blot Protocol for Protein Expression
2
Examining p53 and MDM2 Colocalization in Treated PC12 Cells
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Colocalization of p53 and MDM2 proteins was examined by immunofluorescence staining of the treated PC12 cells. In brief, PC12 cells were cultured on confocal dishes (NEST) at 37 °C for 24 h. After treatments with Aβ25−35, curcumin (5 µM), vorinostat (0.5 µM), and silibinin (1 µM), as described previously, the cells were fixed with 4% PFA for 15 min at room temperature (24 °C), washed twice with PBS, and incubated with 0.1% Triton-X 100 for 15 min at room temperature (24 °C). The cells were then blocked with 1% bovine serum albumin in PBS for 30 min, and subsequently incubated with antibodies against p53 (1:1,000; Cell Signaling Technology) and MDM2 (1:400; Cell Signaling Technology) at 4 °C for 12 h with mild shaking. The cells were washed twice with PBS, then incubated with a secondary antibody at room temperature (24 °C) for 1 h; the secondary antibodies were donkey anti-rabbit 488 (1:1,000; Invitrogen, Camarillo, CA, USA) and donkey anti-mouse 594 (1:1,000; Invitrogen). To stain cell nuclei, the cells were incubated with 1 µg/ml 4′, 6-diamidino-2-phenylindole (DAPI) for 5 min. Next, the cells were washed twice with PBS and observed using a confocal Laser Scanning Biological microscope FV1000 (Olympus).
3
Immunofluorescence Analysis of LC3 in HEK293T Cells
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HEK293T cells (7 × 104) were seeded on glass coverslips (ECN 631–1577; VWR International, Radnor, PA, USA). Cells were washed in PBS, fixed with ice-cold 100% methanol for 10 min, and washed again. Coverslips were blocked for 30 min with PBS buffer with 0.1% Tween-20 containing 3% bovine serum albumin (A 9647; MilliporeSigma) and then incubated overnight at 4°C with primary antibody (LC3 A/B, 4108S; Cell Signaling Technology, Danvers, MA, USA) at 1:200 dilution in 1% bovine serum albumin containing PBS buffer with 0.1% Tween-20. Samples were washed with PBS and then incubated for 1 h at room temperature with Alexa Fluor 488 (4412S; Cell Signaling Technology) secondary antibody at 1:600 dilution. Cells were washed with PBS and treated with DAPI at 1:1000 dilution in PBS for 10 min, followed by wash with PBS. Coverslips were mounted by using FluorSave Reagent (345789; MilliporeSigma) and visualized by using a Zeiss LSM 710 laser confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). Three images per given conditions were taken. All data are expressed as means ± sem . Comparisons between groups were made by using ANOVA with Tukey’s multiple comparison post hoc test. Statistical significance was evaluated, and values of P < 0.05 were considered significant.
4
Gastrocnemius Phospho-eEF2 (Thr 56) Quantification
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For determination of gastrocnemius phospho-eEF2 (Thr 56) protein levels 2 μg/μl gastrocnemius Western blotting preps were made using 4x Laemmli buffer. Thereafter, 30 μl of prepped samples were loaded onto 12% SDS-polyacrylamide gels and subjected to electrophoresis (200 V @ 75 min). Proteins were then transferred to polyvinylidene difluoride membranes (Whatman™, Westran® Clear Signal), and membranes were blocked for 1 h at room temperature with 5% nonfat milk powder.
Rabbit anti-phospho-eEF2 (Thr 56) IgG (1:1,000; Cell Signaling) was incubated with membranes overnight at 4°C in 5% bovine serum albumin (BSA), and the following day membranes were incubated with anti-rabbit or anti-mouse IgG secondary antibodies (1:2,000, Cell Signaling), respectively, at room temperature for 1 h prior to membrane development. Membrane development was performed as described above. As described above, membranes were stained with Coomassie in order to visually ensure between-lane protein loading equality.
Rabbit anti-phospho-eEF2 (Thr 56) IgG (1:1,000; Cell Signaling) was incubated with membranes overnight at 4°C in 5% bovine serum albumin (BSA), and the following day membranes were incubated with anti-rabbit or anti-mouse IgG secondary antibodies (1:2,000, Cell Signaling), respectively, at room temperature for 1 h prior to membrane development. Membrane development was performed as described above. As described above, membranes were stained with Coomassie in order to visually ensure between-lane protein loading equality.
5
Western Blot Analysis of iNOS in LPS and IFNγ Treated Cells
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Whole lysates were prepared from cultures that had been left untreated, treated with LPS (100 ng/mL), or treated with both LPS and IFNγ (250 ng/mL) for 24 hours. Protein concentrations were determined by bicinchoninic acid assay. Protein samples (50 μg per lane) were separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane. After blocking in 5% milk in Tris-buffered saline with 0.1% tween-20 (TBST), membranes were incubated overnight with iNOS (#2977) primary antibody from Cell Signaling Technology (Danvers, MA, USA) diluted 1:500 with 5% bovine serum albumin (BSA) in TBST. After incubation with anti-rabbit IgG horseradish peroxidase-tagged secondary antibody from Santa Cruz Biotechnology (Santa Cruz, CA, USA), bands were visualized by using enhanced chemiluminescence. To normalize protein loading, blots were also probed for β-actin expression.
6
Protein Quantification and Western Blot Analysis
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Total protein in Tris-buffered saline (TBS) and neutralized formic acid (FA) extracts was quantified using the Ready to Use Bradford Reagent (Bio-Rad) and for TBS-Triton X100 (TBSX) extracts the BCA Protein Assay Kit (Pierce).
For western blot analysis of TBS-X fractions protein (20 μg) was separated on 4–12 % Bis-Tris gels (Invitrogen) and transferred onto low-fluorescence PVDF membranes. Membranes were blocked (1 hr. room temperature) with 5 % non-fat milk in TBS containing 0.1 % Tween 20 (TBST), then incubated (4 °C, overnight) with primary antibodies against post synaptic density protein (PSD-95, 1:1000, Cell Signaling), synaptophysin (1:1000, Cell Signaling), the EGF receptor (1:100, Santa Cruz Biotechnology) or actin (1 hr. room temperature, 1: 20,000, Cell Signaling) in TBST with 5 % bovine serum albumin (BSA). Membranes were incubated in secondary antibodies (Jackson Immunoresearch) with TBST wash (3X5min) in between steps and imaged using an Odyssey ® Fc Imaging System. Image J was utilized to quantify actin normalized proteins.
For western blot analysis of TBS-X fractions protein (20 μg) was separated on 4–12 % Bis-Tris gels (Invitrogen) and transferred onto low-fluorescence PVDF membranes. Membranes were blocked (1 hr. room temperature) with 5 % non-fat milk in TBS containing 0.1 % Tween 20 (TBST), then incubated (4 °C, overnight) with primary antibodies against post synaptic density protein (PSD-95, 1:1000, Cell Signaling), synaptophysin (1:1000, Cell Signaling), the EGF receptor (1:100, Santa Cruz Biotechnology) or actin (1 hr. room temperature, 1: 20,000, Cell Signaling) in TBST with 5 % bovine serum albumin (BSA). Membranes were incubated in secondary antibodies (Jackson Immunoresearch) with TBST wash (3X5min) in between steps and imaged using an Odyssey ® Fc Imaging System. Image J was utilized to quantify actin normalized proteins.
7
Immunofluorescence Analysis of SLUG and E-cadherin
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Cells grown on glass coverslips were fixed and permeabilized in phosphate-buffered saline (PBS) containing 2% paraformaldehyde and 1% Triton X-100, while sections were dewaxed, hydrated through decreasing concentrations of ethanol, rinsed in distilled water, and subjected to antigen retrieval treatment. Samples were washed in PBS and then incubated in PBS and 1% bovine serum albumin (Sigma-Aldrich) to block nonspecific binding, before incubating with the primary anti-SLUG (clone L40C6, Cell Signaling Technology) and anti-E-cadherin (clone 32A8, Cell Signaling Technology) antibodies diluted in PBS and 1% bovine serum albumin overnight at 4 °C. Samples were subsequently rinsed in PBS and then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-mouse secondary antibody (Dako). Mounting and nuclei counterstaining were performed using the ‘pro long antifade reagent with DAPI' (Invitrogen, Carlsbad, CA, USA) and observed under a fluorescence microscope (Carl Zeiss, Milan, Italy).
8
Western Blotting of Stimulated Cells
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Cells were plated at a concentration of 3 million cells per 6-well plate and incubated overnight in cell media without FBS. The following day, cells were stimulated with LPS or IL-4 as outlined above. Cell pellets were lysed and 20 μg of lysate was fractionated by SDS-PAGE and transferred to nitrocellulose. Membranes were then blocked with 5% bovine serum albumin, washed, and incubated with primary antibodies (1:2,000) (Cell Signaling Technology, Danvers, MA) overnight at 4 °C. Membranes were then washed and incubated for 1 h with anti-rabbit IgG horseradish peroxidase linked secondary antibody (1:5,000) (ECL, Buckinhamshire, UK) prior to developing with a chemiluminescent system (Amersham, Buckinhamshire, UK). Original immunoblots are shown in Supplementary Fig. 15 .
9
Western Blot Analysis of Cellular Proteins
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Transfected cells were lysed on ice in RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) containing 1 × complete protease inhibitor (Roche, Indianapolis, IN, USA). Debris was pelletized by centrifugation at 13 200 r.p.m. for 15 min, and protein concentrations were determined using the Pierce BCA assay (Thermo Scientific). Lysates were heat-denatured at 100 °C for 10 min before separation in 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose membrane (GE Healthcare, Mickleton, NJ, USA). Membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich) in Tris-buffered saline Tween-20 buffer (10 mM Tris, pH 7.6, 150 mM NaCl and 0.1% Tween-20) and probed with primary antibody in Tris-buffered saline Tween-20 with 5% bovine serum albumin at the recommended dilutions at 4 °C. Primary antibodies included GPRC5A antibody (Sigma-Aldrich), beta-actin antibody (Cell Signaling Technology, Beverly, MA, USA), HuR antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and LSD1 antibody (Cell Signaling Technology). Membranes were incubated with secondary antibody (Cell Signaling Technology) diluted in Tris-buffered saline Tween-20 with 5% bovine serum albumin for 1 gemcitabine at room temperature. The signal was detected with Pierce ECL Western Blotting Substrate (Thermo Scientific) and GE ImageQuant LAS 4000 (GE Healthcare).
10
Lentiviral Transduction Protocol for HEK 293FT Cells
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HEK 293FT cells were plated on six-well or ten-centimeter plates pre-coated with 0.1% (w/v) gelatin (Sigma-Aldrich # G9391). Once cells reached ~90% confluency, they were transfected with 2 μg of lentiviral transfer plasmid, 1.5 μg of psPAX2 (Addgene #12260), and 500 ng of pMD2.G (Addgene #12259) using Lipofectamine 3000 (Invitrogen, Thermo Fisher #L3000001) per the manufacture’s protocol. After 12–18 hours, the media was changed, bovine serum albumin (Cell Signaling Technology #9998) was added to 1% (w/v) along with 2 mM valproic acid (Santa Cruz Biotechnology #sc-202378B) to improve viral gene expression and recovery. After 24 hours, the media was removed from cells and stored at 4°C overnight and replaced again with DMEM plus BSA and VPA. 24–36 hours later media was again harvested and combined with the previous media and concentrated ~5x by centrifugation using 100 kDa MWCO Amicon Ultra-4 (Millipore # UFC810024) or Amicon Ultra-15 (Millipore # UFC910024) centrifugal filter units at 4°C. Concentrated virus was applied through a 0.45 μm surfactant free cellulose acetate syringe filter (Nalgene, Fisher Scientific #09–740-106) and immediately used or stored at −80°C for up to one month. Target cells were transduced in suspension with the filtered virus-containing media in the presence of 8 μg/mL polybrene (Santa Cruz Biotechnology, #sc-134220).
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