Total proteins were extracted from cells using RIPA lysis buffer (Beyotime) supplemented with phenylmethylsulfonyl fluoride (Sigma‐Aldrich). The mixture of cellular homogenate and loading buffer was heated at 95°C for 10 minutes. The samples were run at 100 V on a 10% acrylamide gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Sigma‐Aldrich) at 300 mA for 1 hour. The PVDF membranes were blocked in QuickBlock blocking buffer (Beyotime) and incubated with a primary antibody at 4°C overnight, followed by incubation with an appropriate secondary antibody. The protein bands were visualized using a Clarity Western enhanced chemiluminescence substrate kit (Bio‐Rad) and a ChemiDoc XRS+ gel imaging system (Bio‐Rad). The primary antibodies used were as follows: PTBP1 (ab134950, 1:10000; Abcam), PKM1 (NBP2‐14833SS, 1:2000; Novus), PKM2 (NBP1‐48308SS, 1:2000; Novus), and GAPDH (ab181602, 1:10000; Abcam).
Ab134950
Manufactured by Abcam
Sourced in United Kingdom
Ab134950 is a laboratory reagent for use in research applications. It is a highly specific antibody that recognizes a target antigen. The core function of this product is to provide a tool for researchers to detect and study the target of interest. No further details on the intended use or application are provided.
Automatically generated - may contain errors
Lab products found in correlation
Ecl reagent, by Merck Group (2 mentions)
Lysis buffer, by Beyotime (2 mentions)
Ab213524, by Abcam (2 mentions)
Ab252421, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Chemidoc xrs gel imaging system, by Bio-Rad (1 mentions)
Quickblock blocking buffer, by Beyotime (1 mentions)
Ab181602, by Abcam (1 mentions)
Clarity western enhanced chemiluminescence substrate kit, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab8245, by Abcam (1 mentions)
Ab6276, by Abcam (1 mentions)
Polyvinylidene di uoride membrane, by Merck Group (1 mentions)
3 protocols using ab134950
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Protein extraction was carried out with a lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Proteins were quantified by an acid-based Protein Assay Kit (Thermo), followed by separation with 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transfer to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Afterward, 5% skim milk was used to block (2 h) the non-specific binding sites of membranes. Membranes were subsequently incubated with indicated primary antibodies overnight against PD-L1 (1:1000, ab213524, Abcam, Cambridge, UK), FOSL1 (1:1000, ab252421, Abcam), or PTBP1 (1:5000, ab134950, Abcam), followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:5000, ab205718, Abcam). Membranes were washed with PBS three times, and signals were visualized by an ECL reagent (Millipore, Bedford, MA, USA). Protein expression was analyzed using ImageJ software 6.0 (National Institutes of Health, Bethesda, MD, USA). GAPDH (1:2000, ab8245, Abcam) and β-actin (1:5000, ab6276, Abcam) acted as internal controls.
3
Protein Expression Analysis via Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Protein extraction was carried out with lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Then proteins were quanti ed by an acid-based Protein Assay Kit (Thermo), followed by separate with 10% SDS-PAGE by electrophoresis and transfer to polyvinylidene di uoride membranes (Millipore, Bedford, MA, USA). Afterwards, 5% skim milk (2 h) was used to block the non-speci c binding sites of the membranes. The membranes were subsequently incubated with indicated primary antibodies overnight against PD-L1 (1:1000, ab213524, Abcam, Cambridge, UK), FOSL1 (1:1000, ab252421, Abcam), PTBP1 (1:5000, ab134950, Abcam), followed by incubate with horseradish peroxidase-conjugated secondary antibodies. Washed with PBS for three times and the signals were visualized by ECL reagent (Millipore).
Protein expression was then analyzed by ImageJ software 6.0 (National Institutes of Health, USA). GAPDH and β-actin acted as an internal control.
Protein expression was then analyzed by ImageJ software 6.0 (National Institutes of Health, USA). GAPDH and β-actin acted as an internal control.
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