Cells were collected and washed twice with PBS and then lysed with lysis buffer (Nanjing KeyGen Biotech Co., Ltd.) for 30 min on ice. Xenograft tissues were ground up in liquid nitrogen and lysed with 100–200 µl lysis buffer (Nanjing KeyGen Biotech Co., Ltd.) for 30 min on ice. The proteins were then centrifuged at 11,000 × g for 20 min at 4°C. The concentrations of proteins were detected by BCA kit (Bioworld, Guangzhou, China). Then, 30 µg protein was separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were blocked with 5% BSA for 1 h and incubated with rabbit polyclonal anti-BAG4 (ab2048; 1:100; Abcam), mouse monoclonal anti-α-tubulin (T6199; 1:1,000; Sigma-Aldrich; Merck KGaA) and anti-GAPDH (G8795; 1:1,000; Sigma-Aldrich; Merck KGaA) primary antibodies for overnight at 4°C. The membranes were washed three times for 10 min with PBST (PBS 1,000:Tween-1) and incubated with HRP-conjugated goat anti-rabbit (FDR007; 1:10,000; Fdbio Science, Hangzhou, China) or anti-mouse (FDM007; 1:10,000; Fdbio Science) for 1 h at 37°C. The membranes were then washed three times for 10 min with PBST and visualized with Pico ECL (Fdbio Science) by tanon-5200 (Tanon Science and Technology Co., Ltd., Shanghai, China). α-tubulin and GAPDH served as internal controls.
Lysis buffer
Manufactured by Keygen Biotech
Sourced in China
Lysis buffer is a commonly used solution in molecular biology and biochemistry laboratories. It is designed to disrupt the cell membranes and release the cellular contents, including proteins, nucleic acids, and other biomolecules. The buffer typically contains a combination of detergents, salts, and other components that facilitate the lysis process.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (14 mentions)
Bca protein assay kit, by Keygen Biotech (13 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
β actin, by Abcam (7 mentions)
Quantity one software, by Bio-Rad (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Chemidoc touch imaging system, by Bio-Rad (4 mentions)
Gapdh, by Proteintech (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Bca kit, by Keygen Biotech (3 mentions)
Chemiluminescence kit, by Merck Group (3 mentions)
Zb 2301, by ZSGB-BIO (3 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Keygen Biotech (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Gapdh, by Abcam (2 mentions)
Nitrocellulose membrane, by Promega (2 mentions)
84 protocols using lysis buffer
1
Western Blot Analysis of BAG4 Protein
2
Western Blot Protein Analysis
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Total protein was extracted from cells with lysis buffer (KeyGEN BioTECH, China). Total protein samples were separated by SDS-PAGE (10–15%) and transferred to a PVDF membrane. After blocking with 5% defatted milk for 1 h, the membrane was incubated with primary antibodies overnight at 4 °C and then further incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Protein bands were detected using chemiluminescence (KeyGEN BioTECH, China).
3
Protein extraction and western blotting
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Proteins were extracted from the NSCLC cells (n = 3, biological replicates) or tumor tissues in the lysis buffer (KeyGEN BioTECH, Nanjing, China) and protease inhibitor cocktail (KeyGEN BioTECH, Nanjing, China) for western blotting34 (link). Antibodies used for western blotting were: anti-IRX4 (ab123542, Abcam, Cambridge, UK), anti-pEGFR, EGFR, p-smad3, smad2/3 (#3777, #4267, #9520, #8685, Cell Signaling Technology, Boston, MA, USA), anti-CD133, NANOG, Notch1, β-catenin, MDR1, VDR, ABCG2 and anti-Histone-H3 (18470-1-AP, 14295-1-AP, 20687-1-AP, 51067-2-AP, 22336-1-AP, 14526-1-AP, 27286-1-AP, 17168-1-AP, Proteintech group, WUHAN SANYING, WuHan, China), anti-Sox2, Oct4, ALDH1A1 (D164316, D121072, D220058, Sangon Biotech, Shanghai, China), and anti-β-actin, anti-GAPDH (bs-0061R, bs-0755R, Bioss, Beijing, China) antibodies, HRP-conjugated goat anti-rabbit IgG secondary antibody (ABL3012-2, AbSci, Vancouver, WA, USA). The electrochemical luminescent substrates (Tanon, Shanghai, China) were used according to the manufacturer’s protocol in order to visualize the proteins of interesting in the Tanon imaging system. The relative expressions were quantified densitometrically using the ChemiScope analysis software and calculated according to the reference bands of GAPDH or β-actin.
4
Western Blot Protein Analysis
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Cells were lysed in lysis buffer supplemented with protease and phosphatase inhibitors (Nanjing KeyGen Biotech Co., Ltd.). The Bio-Rad assay kit (Bio-Rad Laboratories, Hercules, CA, United States) was used to determine protein concentrations. Protein electrophoresis was performed on 8%, 10%, and 15% SDS polyacrylamide gels, and proteins were transferred to PVDF membranes, blocked with 5% non-fat dried milk at room temperature for 2 h, and then probed with the appropriate primary antibody overnight at 4°C. After the membranes were washed with 0.1% TBS-T and probed with the corresponding secondary antibody at room temperature for 2 h, protein bands were observed via enhanced chemiluminescence detection.
5
Western Blot Analysis of Renal Fibrosis Markers
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The renal tissue was homogenized in lysis buffer (Keygen Biotech, Nanjing, People’s Republic of China) on ice for 15 minutes. Protein samples at 20 μg were loaded onto the 10% SDS-PAGE gel and then transferred onto polyvinylidenefluoride (PVDF) membranes. Anti-TGF-β1 (1:1,000, Abcam), anti-Smad2 (1:1,000, Abcam), anti-phospho-Ser-467-Smad2 (1:500, Abcam), anti-CTGF (1:500, Abcam), and anti-GAPDH (1:1,000, Abcam) antibodies were used to incubated with the membranes overnight at 4°C. Then horseradish peroxidase-conjugated secondary antibodies incubated the membranes for 1 hour at room temperature. Enhanced chemiluminescence regents (Immobilon Western, EMD Millipore, Billerica, MA, USA) were used, and data were collected by C-digit machine (LI-COR Biosciences, Cambridge, UK).
6
Quantitative Protein Analysis from C6/36 Cells
7
Western Blot Analysis of ATXN2L, pAKT, and GAPDH
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For Western blot, antibodies of ATXN2L (Proteintech, Rosemont, IL), pAKT (Cell Signaling Tech., Danvers, MA, US), and GAPDH (Proteintech) were used. Procedures was as previously described21 (link). Generally, cells or tissues were solubilized in lysis buffer (KeyGEN, Nanjing, China), and total protein concentration was determined by BioRad BCA method (Pierce, Rockford, IL, USA). After electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gels, protein was electroblotted onto polyvinylidene fluoride membranes. Then, membranes were blocked with 5% bovine serum albumin and incubated overnight at 4 °C with antibodies against ATXN2L, pAKT, or GAPDH, respectively. After incubated by secondary antibodies, blots were visualized using enhanced chemiluminescence.
8
Western Blot Analysis of Akt and Ezrin
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CRC cells were lysed on ice by lysis buffer (#KGP701, KeyGen Biotech, China) followed by quantificational measure with a bicinchoninic acid (BCA) Protein Assay Kit (#KGPBCA, KeyGen Biotech, China). Then, the lysates were subjected to SDS-polyacrylamide gel electrophoresis for separation. The isolated proteins were transferred onto polyvinylidene fluoride (PVDF) membrane (#IPVH00010, Millipore, USA). After fixing in methanol for 1 min, the PVDF membranes were blocked in PBST solution containing 5% nonfat milk and subsequently incubated overnight at 4°C with the following primary antibodies, respectively: anti-Akt (1:1,000 dilution, #YT0178, immunoway, USA), anti-p-Akt (Ser-473) (1:1,000, #YP0006, immunoway, USA), anti-Ezrin (1:5,000 dilution, #26056-1-AP, proteintech, USA), and anti-β-tubulin (1:5,000 dilution, #10094-1-AP, proteintech, USA). Then, the PVDF membranes were incubated with the secondary antibodies (Goat anti-rabbit IgG, 1:5,000 dilution, #BS13278, Bioworld, USA; Goat anti-mouse IgG, 1:5,000 dilution, #BS12478, Bioworld, USA) and were visualized with FDbio-Dura ECL Kit (#FD8020, Fdbio, China). All experiments were repeated three times and quantified using Gel-pro software.
9
Protein Expression Analysis in Colorectal Cancer
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Proteins from colorectal cancer cell lines were extracted with lysis buffer follow instructions (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The BCA Protein Assay kit (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was applied to detect the protein concentration. Protein lysate was separated on SDS-PAGE gels and transferred subsequently onto polyvinylidene fluoride membranes (EMD Millipore). TBS containing 0.1% Tween-20 was performed to block the membrane. Then, membranes were incubated with rabbit anti Homer1 monoclonal antibody (ab184955; Abcam, Cambridge, MA, USA), G3BP1 (ab181150; Abcam, Cambridge, MA, USA) or GAPDH (cat. no. T0004; 1:5000; Affinity Biosciences) overnight, and were cultured with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies (1:5000; cat. no. SA00001-2; ProteinTech Group, Inc., Chicago, IL, USA) for 2 h at 37°C. Enhanced chemiluminescence (ECL) reagents (cat. no. KGP1127; Nanjing KeyGen Biotech Co., Ltd) were utilized to reveal protein bands. The protein levels were normalized to the GAPDH protein levels.
10
Western Blot Analysis of ZEB1 and E-cadherin
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Cells were lysed with ice-cold lysis buffer (Keygen Biotech, China) for 30 min on ice. Cell lysates were then collected after centrifugation at 12,000 rpm for 5 min at 4 °C. The lysate protein at an amount of 60 µg was loaded, and the total cellular protein was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transblotted overnight at 4 °C onto polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with antibody (anti-ZEB1, 1:1,000 dilution, Cell Signaling Technology (CST), USA; anti-E-cadherin, 1:2,000 dilution, CST, USA) at 4 °C overnight, washed three times with tris-buffered saline supplemented with 0.1% tween (TBST), and incubated with the IRDye® 800CW Conjugated Goat anti-Rabbit IgG (Catalog Number 926-32211, LI-COR Biosciences, USA) secondary antibody (1:2000 dilution) for 1 h at room temperature. The membranes were washed three times with TBST, and the immunoreactive bands were detected using the kit and the infrared (IR) laser-based instrumentation from LI-COR Biosciences, USA.
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