Western blot analysis were conducted following standard protocols as described earlier [42 (link)]. Briefly, whole cell lysates were prepared and protein concentrations were quantified colourimetrically using a BCA Protein Assay Kit (Solarbio, Beijing). Samples were separated in a 10% SDS-polyacrylamide gel (Solarbio, Beijing) and blotted onto a PVDF membrane (Millipore). Immunoblot was performed with commercially available antibodies (anti PTEN antibody, 1:5000, Santa Cruz; anti- AKT antibody, 1:1000, BD Bioscience; anti-P-AKT antibody, 1:1000, Cell Signaling Technology; anti-GAPDH antibody, 1:5000, Santa Cruz.). ECL (Millipore) was applied for chemiluminescence detection. Immunoblot signal quantifications were performed using Image J software.
Sds polyacrylamide gel
Manufactured by Solarbio
Sourced in China
The 10% SDS-polyacrylamide gel is a laboratory equipment used for the separation and analysis of proteins based on their molecular weight. It is a common tool in biochemistry and molecular biology research.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Anti gapdh antibody, by Santa Cruz Biotechnology (3 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (3 mentions)
Ripa buffer, by Beyotime (2 mentions)
Anti pten antibody, by Santa Cruz Biotechnology (1 mentions)
Anti p akt antibody, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
Bca protein detection kit, by Solarbio (1 mentions)
Anti n cadherin, by Abcam (1 mentions)
Anti β catenin, by Abcam (1 mentions)
Bca kit, by Solarbio (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Goat anti mouse igg h l hrp, by Abcam (1 mentions)
Ab6789, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
5 protocols using sds polyacrylamide gel
1
Quantitative Western Blot Analysis
2
Western Blot Analysis of STAMBPL1 Expression
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Western blot analysis was performed according to standard protocols. Briefly, whole cell lysates were prepared, and the protein concentration was colorimetrically quantified using the BCA protein detection kit (Beijing Solarbio). The samples were separated in 10% SDS polyacrylamide gel (Solarbio, Beijing) and blotted on PVDF membrane (Millipore). Immunoblotting was performed using commercially available antibodies (anti-STAMBPL1 antibody, 1:5000, Santa Cruz; anti-GAPDH antibody, 1:5000, Santa Cruz.). Capillary electrophoresis (ECL) was used for chemiluminescence detection. Image J software was used to quantify the immunoblot signal.
3
Western Blot Protein Quantification
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Cells were washed twice with PBS, and total protein was extracted using RIPA buffer (Beyotime Biotechnology). Protein concentration was determined using a BCA Protein Assay Kit (Beyond Biotechnology). Protein samples were separated using 10% SDS‐polyacrylamide gel (Solarbio) after denaturation. Then, proteins were transferred to a PVDF membrane (Millipore) which were blocked with 5% skimmed milk for 1 h. The blots were incubated overnight with the first antibodies (anti MITD1 antibody, 1:5000, Santa Cruz; anti‐GAPDH antibody, 1:5000; Santa Cruz). ECL (Millipore) After incubation with secondary antibodies, an enhanced chemiluminescence reagent was applied for detection.
4
Western Blot Analysis of EMT Markers
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Western blot analysis was performed as previously reported23 (link). In brief, cell lysates were prepared with RIPA buffer (Beyotime Biotechnology, Shanghai, China). Next, BCA Kit (Solarbio, Beijing) was used to quantify the protein concentrations. Samples were segregated by 10% SDS-polyacrylamide gel (Solarbio, Beijing) and transferred onto a PVDF membrane (Millipore). Subsequently, the immunoblot was incubated with anti-E-cadherin (1:1000, ab76055), anti-β-catenin (1:1000, ab32572), anti-N-cadherin (1:1000, ab76057), anti-Vimentin (1:1000, ab8978), anti-GAPDH (1:1000, ab8245) at 4 °C overnight and with Goat Anti-Mouse IgG H&L (HRP, 1:2000, ab6789) at 37 °C for 1 h. The primary and secondary antibodies were purchased from Abcam (Cambridge, UK). Next, ECL (Millipore) was applied for chemiluminescence detection. The immunoblot signal was quantified with Image J software.
5
Western Blot Analysis of YY1 and CDK6
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HEK293T and PC-3 cells were lysed, proteins were extracted and western blotting was performed, as previously described (13 (link)). Equal amounts of cell lysates (15 µg) were fractionated by size on a 10% SDS-polyacrylamide gel (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and transferred onto polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membrane was blocked with tris-buffered saline-Tween with 5% non-fat milk at room temperature for 1 h. Samples were then incubated with antibodies against the following proteins: YY1 (CST-2185S; 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CDK6 (D155263; 1:1,000; Sangon Biotech Co., Ltd., Shanghai, China) and β-actin (MABT825; 1:10,000; EMD Millipore) at 4°C overnight. Subsequently, horseradish peroxidase-labeled goat anti-rabbit (ab6721; 1:10,000; Abcam, Cambridge, MA, USA) and goat anti-mouse (ab6789; 1:10,000; Abcam) secondary polyclonal antibodies were added, respectively, followed by immobilon enhanced chemiluminescence (EMD Millipore).
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