Cells or tissue samples were collected and lysed in RIPA buffer containing proteinase inhibitors (Sigma, USA) and phosphatase inhibitors (CWBio, China). The protein extracts were separated on SDS-PAGE gels, followed by electrotransfering onto polyvinylidene fluoride membranes (Sigma, USA). After blocking with 5% fat-free milk for 2 h at room temperature, the membranes were incubated with primary antibodies and subsequently incubated with secondary antibody (Thermo Scientific, MA, USA). Finally bands were scanned with Bio-Rad Imager and individual band intensity was quantified with software of ImageJ. Antibodies were used as follows: caspase 3 (CST, USA), cleaved caspase 3 (CST, USA), GSDMC (Abclonal, China), GSDMD (Abclonal, China), GSDME (Abclonal, China), PI3K (Bioss, China), p-PI3K (Bioss, China), Akt (Proteintech, China), p-Akt (CST, USA), Bcl-2 (Proteintech, China), Bax (Proteintech, China) and β-Actin (Abclonal, China).
Gsdme
Manufactured by ABclonal
Sourced in China
GSDME is a lab equipment product designed for use in scientific research. It serves as a tool for cell manipulation and analysis. The core function of GSDME is to enable researchers to perform various cell-related experiments and investigations. No further details or interpretation on the intended use of this product are provided.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Proteinase inhibitor, by Merck Group (1 mentions)
Phosphatase inhibitor, by CWBIO (1 mentions)
P pi3k, by Bioss Antibodies (1 mentions)
β actin, by ABclonal (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Bcl 2, by Proteintech (1 mentions)
Gsdmd, by ABclonal (1 mentions)
Gsdmd n, by Affinity Biosciences (1 mentions)
Cleaved caspase 8, by Affinity Biosciences (1 mentions)
Hrp conjugated secondary antibodies, by Wanlei (1 mentions)
Gsdmd, by Affinity Biosciences (1 mentions)
Cleaved caspase 1, by Wanlei (1 mentions)
Sds page, by Wanlei (1 mentions)
Cleaved caspase 3, by Wanlei (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Gsdme n, by Abcam (1 mentions)
β actin, by Huabio (1 mentions)
3 protocols using gsdme
1
Western Blot Analysis of Apoptosis Markers
2
Pyroptosis Pathway Protein Detection
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The cells were harvested in RIPA Lysis Buffer and lysed using ultrasound (Wanleibio, Shenyang, China). BCA Reagent was used to determine total protein content (Wanleibio, Shenyang, China). SDS-PAGE (Wanleibio, Shenyang, China) was used to separate equivalent quantities of protein extract, which was then deposited onto PVDF membranes (Millipore, USA). Cleaved-Caspase-1 (Wanleibio, Shenyang, China), cleaved-Caspase-3 (Wanleibio, Shenyang, China), cleaved-Caspase-4 (Affinity Biosciences, Suzhou, China), cleaved-Caspase-8 (Affinity Biosciences, Suzhou, China), GSDMD (Affinity Biosciences, Suzhou, China), GSDME (ABclonal, Wuhan, China), and GSDMD-N (Affinity Biosciences, Suzhou, China) were the primary antibodies employed in this test. After blocking with 5% skim milk for an hour, the membranes were incubated overnight with primary antibodies at 4°C. The membranes were then incubated with HRP-conjugated secondary antibodies (Wanleibio, Shenyang, China) and detected using an enhanced chemiluminescence substrate kit (Wanleibio, Shenyang, China) after washing.
3
Western Blot Analysis of Protein Expression
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Total protein from the above tumor tissues, MC38 cells and CT26 cells treated with TBMS1 and TEPP-46, RAW264.7 induced by 20 ng/mL IL-4 and treated with TBMS1 was extracted by using RIPA lysis (Millipore, USA) added proteinase and phosphatase inhibitor cocktails (Solarbio, Beijing). The protein concentration was determined with the Pierce BCA detection kit (Thermo Fisher Scientific, USA). The sample with 20-80 µg protein was electrophoresed by 10% polyacrylamide gradient gels at 110 v for 1.5 h and transferred onto 0.45 µm PVDF membrane (Millipore, United States), then TBST containing 5% skim milk was used to block membranes for 2 h. The treated membranes could be incubated with primary antibodies including PKM2 (Huabio, CHN), caspase-3 (Elabscience, CHN), cleaved-caspase-3 (Cell Signaling Technology, USA), GSDME (Abclonal, CHN), GSDME-N (Abcam, UK), STAT6 (Absin, CHN), p-STAT6 (Absin, CHN), β-actin (Huabio, CHN). After incubating overnight at 4°C, the membranes were washed with TBST, and the anti-rabbit or antimouse HRP-conjugated secondary antibody (Bioss, CHN) was incubated with the membranes for 2 h at room temperature. After being washed again by TBST, band detection was visualized by enhanced chemiluminescence reagent (Millipore, USA). Finally, Image J software was used to quantify the integrated density of the target proteins.
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