Cell samples were washed thrice with precooled PBS and lysed in SDS buffer (Beyotime, Shanghai, China) containing 1 mM PMSF (Beyotime). Lysates were heated at 100°C for 15 min and centrifuged at 12,000 × g for 15 min at 4°C. The protein concentrations were then measured with a bicinchoninic acid (BCA) protein assay kit (Beyotime), and the cell lysates were mixed with 5 × SDS-PAGE buffer (P0015, Beyotime, Shanghai, China) and boiled for 10 min. Subsequently, samples were subjected to 10–12% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, United States). The PVDF membranes were incubated with interesting primary antibodies overnight at 4°C after blocking with 5% skimmed milk for 1 h, then indicated with appropriate HRP-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, United States) for 1 h at room temperature. Finally, target proteins were visualized by enhanced chemiluminescence.
Sds page buffer
Manufactured by Beyotime
Sourced in China
SDS-PAGE buffer is a solution used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to separate proteins based on their molecular weight. It consists of a buffer system and a denaturing agent, which helps to unfold the proteins and give them a uniform negative charge.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (3 mentions)
Sds buffer, by Beyotime (2 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Np 40 lysis buffer, by Beyotime (1 mentions)
Ras antibody, by Cell Signaling Technology (1 mentions)
Gapdh mouse monoclonal antibody, by Proteintech (1 mentions)
Phospho akt ser473 d9e xp rabbit mab, by Cell Signaling Technology (1 mentions)
Tgrinder, by Tiangen Biotech (1 mentions)
Phospho p44 42 mapk erk1 2 thr202 tyr204 d13.14.4e, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Enhanced chemiluminescence detection kit, by Beyotime (1 mentions)
Bca reagent, by Applygen (1 mentions)
Gapdh, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
β tubulin, by ABclonal (1 mentions)
Quantify one software, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
6 protocols using sds page buffer
1
Western Blot Sample Preparation
2
Protein Extraction and Western Blot Analysis
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The tissue samples were homogenized at low temperature, after which NP40 lysis buffer (Beyotime, Shanghai, China) was used to extract protein. The concentration of extracted protein was determined by the BCA Protein Assay Kit (Beyotime, Shanghai, China). Subsequently, the same amounts of protein in cell lysate were divided by SDS-PAGE buffer (Beyotime, Shanghai, China), and then transferred onto a nitrocellulose membrane. The different membranes were immunoblotted with different primary antibodies at 4 °C for 12–18 h. After that, the membranes were immunoblotted with the corresponding secondary antibody at 37 °C for 1 h. The blots were scanned through the Odyssey Infrared Imaging System (LI-COR, Nebraska, USA). When analyzing the results, Image J software was used to analyze the gray value of the protein band, calculate the ratio of the gray value of the target protein to the GAPDH protein, and then normalize the ratio of the different experimental groups.
3
Cellular Signaling Pathway Analysis
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A549 and H2228 cells were plated in six-well plates at a confluency of 70%. 48 hours after adenovirus infection, whole-cell extracts were prepared by lysing cells with the addition of 500 µL of hot SDS-PAGE buffer (Beyotime, P0015B). Tumor tissues were homogenized by TGrinder (Tiangen, OSE-Y30), and lysed with RIPA buffer containing complete protease inhibitor cocktail (Roche, 11697498001). Target proteins were detected by western blot analysis with the following antibodies: GAPDH mouse monoclonal antibody (Proteintech, 60004-1-Ig,), Akt (pan) (40D4) mouse monoclonal antibody (Cell Signaling Technology, 2920), Phospho-Akt (Ser473) (D9E) XP Rabbit mAb (Cell Signaling Technology, 4060), p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb (Cell Signaling Technology, 4695), Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) (Cell Signaling Technology, 4370), mouse monoclonal Anti-MAP Kinase, activated (Diphosphorylated ERK-1&2) antibody (Sigma-Aldrich, M8159), Ras Antibody (Cell Signaling Technology, 3965), and Anti-RAS (G12S) Mouse Monoclonal Antibody (NewEast Biosciences, 26186).
4
Protein Extraction and Western Blot Analysis
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NSCLC cells were lysed in lysis buffer (RIPA buffer and 1% PMSF) (Beyotime) on
ice. Then, the cell lysate was sonicated for 15 s and centrifuged at 12 000
r/min for 15 min. The protein concentrations were quantified using the BCA
reagent (Applygen Technologies, Beijing, China). The whole-cell extracts were
boiled for 5 min in sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE) buffer (Beyotime). The samples were separated in 8–12%
SDS–polyacrylamide gels and electrotransferred to polyvinylidene difluoride
membranes. After blocking with 8% non-fat milk in Tris-buffered saline Tween,
the membranes were probed overnight with the following antibodies at 4°C: GAPDH
(Abcam), β-tubulin (ABclonal, Seoul, Korea), β-actin (Abcam), and KRT8
(ABclonal). The membranes were then incubated with horseradish
peroxidase-conjugated goat anti-rabbit and goat anti-mouse (Abcam) secondary
antibodies at RT for 1 h. Finally, an enhanced chemiluminescence detection kit
(Beyotime) was used to visualize the immunoreactive proteins.7 (link)
ice. Then, the cell lysate was sonicated for 15 s and centrifuged at 12 000
r/min for 15 min. The protein concentrations were quantified using the BCA
reagent (Applygen Technologies, Beijing, China). The whole-cell extracts were
boiled for 5 min in sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(SDS-PAGE) buffer (Beyotime). The samples were separated in 8–12%
SDS–polyacrylamide gels and electrotransferred to polyvinylidene difluoride
membranes. After blocking with 8% non-fat milk in Tris-buffered saline Tween,
the membranes were probed overnight with the following antibodies at 4°C: GAPDH
(Abcam), β-tubulin (ABclonal, Seoul, Korea), β-actin (Abcam), and KRT8
(ABclonal). The membranes were then incubated with horseradish
peroxidase-conjugated goat anti-rabbit and goat anti-mouse (Abcam) secondary
antibodies at RT for 1 h. Finally, an enhanced chemiluminescence detection kit
(Beyotime) was used to visualize the immunoreactive proteins.7 (link)
5
Protein Extraction and Western Blot
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Cell samples were rinsed thrice with precooled PBS and lysed in SDS buffer (Beyotime) containing 1 mM PMSF (Beyotime). Lysates were heated at 100°C for 15 min. Insoluble cell debris was discarded following centrifugation at 12,000 × g for 10 min at 4°C. The protein concentrations of retained supernatant was measured using an enhanced bicinchoninic acid (BCA) protein assay kit (Beyotime). Subsequently, the lysates were mixed with calculated volumes of 5× SDS-PAGE buffer (Beyotime) and SDS buffer to obtain equivalent protein concentrations and then boiled for 10 min. Finally, samples were separated using 8-10% gradient SDS-PAGE, transferred to polyvinylidene fluoride membranes (Merck Millipore, Darmstadt, Germany), and probed with indicated primary antibodies and then with HRP-conjugated secondary antibodies (Invitrogen). Immunoreactive bands were detected using enhanced chemiluminescence (Millipore), and the band intensity of each target protein was calculated using Quantify One software (Bio-Rad, Hercules, CA, USA) and normalized to GAPDH band intensity.
6
Western Blot Analysis of Protein Expression
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The tissue was lysed in RIPA buffer (Beyotime, China), containing 1 mm PMSF for 30 min at 4 °C, and centrifuged at 12,000× g for 15 min at 4 °C. The tissue lysate was mixed with 5× SDS-PAGE buffer (Beyotime) and boiled for 10 min. Then, 30 μg of each sample was subjected to 10% SDS-PAGE and Western blotting, as previously reported [24 (link)]. The primary antibody diluted 1:1000 and secondary antibody diluted 1:2000 in 0.5% BSA-TBST. Following a further four 10 min washes in TBST, the membranes were detected with 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)/nitroblue tetrazolium (NBT) substrate (Sangon Biotech, Shanghai, China). Each experiment was performed in triplicate.
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