After the experiment, the cells were collected, digested with 0.25% trypsin, collected into EP tubes, and protease inhibitors were added. The whole protein extraction kit (BC3710, Solarbio Life Sciences, Beijing, China) obtained the cell protein solution, and the BCA method (PC0020, Solarbio Life Sciences, Beijing, China) was used to detect the protein concentration. SDS-PAGE electrophoresis was performed, the protein was transferred to PVDF membrane and it was blocked with 5% nonfat milk powder for 1 h at room temperature, GAPDH (anti-GAPDH (MA1-16757, Invitrogen) was used as the normalizing antibody, and the corresponding primary antibodies (anti-NLRP3 (MA5-23919, Invitrogen), anti-ASC (PA5-83948, Invitrogen), anti-Caspase-1 (2225S, CST), and anti-GSDMD (39754S, CST)) were added and incubated at 4°C overnight. Horseradish peroxidase-labeled secondary antibody was used, and ECL color was developed, protein ladders are used to mark target protein positions (26617, thermo scientific). The gray value of the target band was analyzed with a gel image processing system (Image J software).
Anti gapdh ma1 16757
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-GAPDH (MA1-16757) is a laboratory product from Thermo Fisher Scientific. It is an antibody that specifically recognizes the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein. GAPDH is a commonly used reference or housekeeping gene in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Bc3710, by Solarbio (1 mentions)
Ma5 23919, by Thermo Fisher Scientific (1 mentions)
Pc0020, by Solarbio (1 mentions)
Bca protein assay kit, by Santa Cruz Biotechnology (1 mentions)
Anti gpr27 pa5 110977, by Thermo Fisher Scientific (1 mentions)
Ecl western blotting substrate kit, by Abcam (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
2 protocols using anti gapdh ma1 16757
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of GPR27 Expression
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Protein expression levels were assessed through Western blot analysis. Proteins were extracted from the U87 and U251 cell lines utilizing RIPA lysis buffer, and their concentrations were subsequently semi-quantified using a BCA protein assay kit provided by Santa Cruz Biotechnology (Dallas, TX, USA). Proteins, in the amount of 20 µg per lane, were resolved via 12% SDS-PAGE and subsequently transferred onto PVDF membranes. These membranes were then blocked using 5% BSA sourced from Sigma-Aldrich for 1 h at ambient temperature before the blocking solution was discarded. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies: anti-GPR27 (PA5-110977, Invitrogen, Cambridge, MA, USA) and anti-GAPDH (MA1-16757; Invitrogen, Cambridge, MA, USA), both diluted at a ratio of 1:2000. Following the primary antibody incubation, the membranes were washed with TBST containing 0.1% Tween. Then, an HRP-conjugated secondary antibody was applied to incubate at room temperature for 1 h. Detection of the proteins of interest was achieved using the ECL Western Blotting substrate kit (Cattegory #ab65623; Abcam). Each experiment was repeated three times independently.
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