For the extraction of total proteins, Western cell lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and other protease inhibitors including cocktail (MedChemExpress, Monmouth Junction, NJ, United States), and phosStop (Roche; Basel, Switzerland) was used. Briefly, cells were lysed with the lysis buffer for 20 min in ice and centrifuged at 14,000 g at 4°C for 20 min as described previously (Liu et al., 2021 (link)). Followed by the collection of supernatants, total concentration of protein was assessed by BCA protein kit assay and equal amount of each sample were subjected for separation through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to PVDF membranes using wet transfer system. Further, 5% skimmed milk was used for the blockage of membranes for 2 h at room temperature and then primary antibodies; anti-p-JAK2, anti-JAK2, anti-p-STAT3, anti-STAT3, anti-PCNA or anti-GAPDH (dilution factor 1:1000) were incubated at 4°C for overnight. After that, membranes were washed thrice with TBST buffer and membranes were incubated with anti-rabbit or mouse secondary antibody conjugated to horse radish peroxidase (dilution factor 1:5000) for 1 h at room temperature. The protein expression was detected by chemiluminescence kit and ImageJ Software.
Western cell lysis buffer
Manufactured by Beyotime
Sourced in China
Western cell lysis buffer is a solution used to extract and solubilize proteins from cells for Western blotting analysis. It contains a mixture of detergents, salts, and buffers that help to disrupt cell membranes and release the cellular contents, including the proteins of interest.
Automatically generated - may contain errors
Lab products found in correlation
Image lab analysis software, by Bio-Rad (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca assay kit, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phosstop, by Roche (1 mentions)
Ecl chromogenic substrate, by GE Healthcare (1 mentions)
Gapdh, by Merck Group (1 mentions)
Ab32089, by Abcam (1 mentions)
Sab4500797, by Merck Group (1 mentions)
Horseradish peroxidase hrp labeled secondary antibody, by Merck Group (1 mentions)
Ecl detection system, by GE Healthcare (1 mentions)
β actin, by Abcam (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab238099, by Abcam (1 mentions)
Ab237704, by Abcam (1 mentions)
Ab280088, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Enhanced chemiluminescence, by Cell Signaling Technology (1 mentions)
P akt ser473 4060, by Cell Signaling Technology (1 mentions)
6 protocols using western cell lysis buffer
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Glucose Metabolism Proteins
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Transfected SW480 and SW620 cells were washed with PBS and lysed using Western cell lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined by the BCA assay kit (Bio-Rad Laboratories, Inc.). A total of 20 µg protein samples were separated using 10% SDS-PAGE and transferred to PVDF membranes (EMD Millipore) followed by blocking with 5% non-fat milk at 37°C for 1 h. Subsequently, the membranes were probed with primary antibodies against glucose transporter 1 (GLUT1; cat. no. ab40084; 1:5,000; Abcam), lactate dehydrogenase A (LDHA; cat. no. ab226016; 1:8,000; Abcam), CKS1B (cat. no. SAB1408846; 1:8,000; Sigma-Aldrich; Merck KGaA) and GAPDH (cat. no. ab37168; 1:20,000; Abcam) at 4°C overnight. The membranes were probed with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. ab205718; dilution of 1:5,000; Abcam) at 37°C for 2 h after washing three times with TBS + 0.1% Tween-20 (Sangon Biotech Co., Ltd.). The immunoblot was visualized using an enhanced chemiluminescence (ECL) chromogenic substrate (GE Healthcare). Image Lab analysis software (V4.0; Bio-Rad Laboratories, Inc.) was used to analyze the intensities of protein bands.
3
Western Blot Analysis of PI3K/AKT Pathway
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After transfection for 24 hours, Hs 766T or SW1990 cells were lysed by Western cell lysis buffer (Beyotime, Haimen, People's Republic of China). An equal amount of protein samples was loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and blotted onto polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was incubated with 5% skimmed milk for 1 hour at room temperature for blocking. Primary antibodies against p-AKT (07–1398; Sigma), AKT (SAB4500797; Sigma), p-PI3K (ab389562; Abcam, Cambridge, MA, USA), PI3K (ab32089; Abcam), SLC7A11 (SAB2500951; Sigma) and β-actin (A1978; Abcam) were incubated with the membrane at 4°C overnight. Afterwards, horseradish-peroxidase (HRP)-labeled secondary antibody (Sigma) was incubated with the membrane for 2 hours at room temperature. Immunochemical detection was conducted with the enhanced chemiluminescence (ECL) Detection System (GE Healthcare, Chicago, IL, USA).
4
Quantitative Western Blot Analysis of TAB2
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Western cell lysis buffer (Beyotime Institute of Biotechnology) was used to disrupt A549 cells, and the proteins were quantified using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). The quantified protein samples (25 µg) were separated on the 5% stacking and 12% separating gels, and subsequently transferred to a polyvinylidene fluoride membrane. The membrane was blocked using 5% skimmed milk for 1 h at room temperature. The membrane was incubated with anti-TAB2 at the dilution of 1:5,000 (cat. no. ab172412; Abcam) and anti-β-actin at the dilution of 1:20,000 (cat. no. ab115777; Abcam) at 4°C overnight, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (cat. no. ab205718; Abcam) at the dilution of 1:5,000 for 2 h at room temperature. Several X-ray films were used to visualize the protein signal through Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.). Image Lab analysis software (v4.0; Bio-Rad Laboratories, Inc.) was utilized to evaluate the intensities of protein bands.
5
Evaluating Protein Expression in NSCLC
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Non‐small cell lung cancernon‐small cell lung cancer cells were disrupted with Western cell lysis buffer (Beyotime), and the concentrations of protein samples were determined using the bicinchoninic acid (BCA) kit (Pierce Biotech). After separating by 10% separating gel, the protein samples were transferred onto the polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was sealed with 5% non‐fat milk and labeled with primary antibodies (Abcam) overnight at 4℃, including anti‐proliferating cell nuclear antigen (anti‐PCNA, ab280088, dilution of 1:1000), anti‐E‐cadherin (ab238099, dilution of 1:3000), anti‐Bcl‐2 associated X, apoptosis regulator (anti‐Bax, ab32503, dilution of 1:8000), anti‐SLC1A5 (ab237704, dilution of 1:1000), and anti‐GAPDH (ab8245, dilution of 1:10000). The membrane was then incubated with the secondary antibody (Abcam) for 2 h at room temperature. The protein signals were analyzed using the enhanced chemiluminescence reagents (Pierce Biotech). Image Lab analysis software (Bio‐Rad) was used to analyze the intensities of protein bands.
6
Western Blot Analysis of MET, AKT, and p-AKT
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Western blot assay PTC cells were disrupted using Western cell lysis buffer (Beyotime, Shanghai, China). BCA assay kit (Bio-Rad, Hercules, CA, USA) was used to measure the concentrations of protein samples. Proteins (30 μg/lane) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and electro-transferred onto polyvinylidene fluoride (PVDF) membrane (Bio-Rad). After sealing with 5% skim milk for 1 h at 37°C, the membrane was then probed with antibodies against MET (ab51067; Abcam, Cambridge, MA, USA) at the dilution of 1:8,000, AKT (ab18785; Abcam) at the dilution of 1:10,000, phosphorylated AKT (p-AKT; Ser473; #4060; Cell Signaling Technology, Danvers, MA, USA) at the dilution of 1:5,000 and GAPDH (ab9485; Abcam) at the dilution of 1:20,000 overnight at 4°C. Relative enrichment of GAPDH was used as the control. Secondary antibody conjugated with horse radish peroxidase (HRP; Abcam) was then incubated with the membrane for 1 h. Immune blots were measured using enhanced chemiluminescence (Cell Signaling Technology). The intensities of protein bands were analyzed by Image Lab analysis software (V4.0; Bio-Rad).
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