Western blotting analysis was performed as previously described 12 (link). Briefly, total proteins that were extracted using a Total Cell Protein Extraction Kit (KeyGen Biotechnology, Nanjing, China) were divided into equal amounts and electrophoresed, transferred onto a nitrocellulose membrane, and blocked with 2% bovine serum albumin. Primary antibodies against MTBP (1:1000; ab115529, Abcam, Cambridge, UK), MDM2 (1:2000; ab16895), p53 (1:1000; ab131442), p21 (1:2000; ab109520), PUMA (1:2000; ab33906), active caspase3 (1:1000; ab2302), and c-myc (1:1000; ab56) were used to detect the expression of these proteins. After washing four times with TBST/0.1% Tween-20, the membranes were incubated with the corresponding secondary antibody. Bands were visualized using a chemiluminescence kit (Beyotime Biotechnology, Beijing, China) and quantified using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Ab115529
Manufactured by Abcam
Sourced in United Kingdom
Ab115529 is a laboratory equipment product. It serves a core function, but a detailed description while maintaining an unbiased and factual approach is not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab131442, by Abcam (1 mentions)
Ab33906, by Abcam (1 mentions)
Ab109520, by Abcam (1 mentions)
Total cell protein extraction kit, by Keygen Biotech (1 mentions)
Ab2302, by Abcam (1 mentions)
Chemiluminescence kit, by Beyotime (1 mentions)
Ab51037, by Abcam (1 mentions)
Ab138222, by Abcam (1 mentions)
Ab222782, by Abcam (1 mentions)
Sds page gel, by Beyotime (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Bovine serum albumin (bsa), by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
β actin, by Abcam (1 mentions)
2 protocols using ab115529
1
Western Blot Analysis of Key Proteins
2
Protein Extraction and Western Blot Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
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RIPA buffer (Beyotime, China) was used for the collection of protein samples. BCA (Beyotime, China) was used for the determination of the concentration of these protein samples. Next, SDS-PAGE gel (Beyotime, China) was used for the separation of these proteins. And the PVDF membranes (Millipore, USA) were used for the adsorption of these proteins. Next, these membranes were blocked with the BSA (Beyotime, China) for 2 h. After that, the primary antibodies were incubated with these membranes at 4°C overnight. The primary antibodies applied in this were MTBP (ABCAm, ab115529), ZEB2 (ABCAm, ab138222), CD44 (ABCAm, ab51037), CD133 (ABCAm, ab222782) and β-actin (ABCAm, ab8227). All these primary antibodies were diluted with the BSA (Beyotime, China) with the ratio (1:1000). These membranes were incubated with the second antibodies for 2 h in the second day. The second antibodies were also diluted with the BSA with the ratio (1:2000). Finally, immunoreactive signals were determined with the Pierce Western Blotting Substrate (Millipore, USA). The bands were quantified with the ImageJ software (National Institutes of Health, USA).
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