Cell total protein extraction kit (KGP2100, KeyGEN BioTECH, Nanjing, China) was used to extract total cellular proteins, and the protein concentration was measured using the BCA Detection Assay Kit (KGP902, KeyGEN BioTECH, Nanjing, China) according to the manufacturer's instructions. An equal amount (20 μg) of the extracted proteins were subjected to electrophoresis on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to a PDVF membrane (Millipore, Billerica, Mass). Membranes were blocked in TBST containing 5% non-fat milk for 1 h and incubated with primary antibody overnight. Antibodies used in the experiment included anti-TRPC6 antibody (ab62461), anti-PCNA antibody (ab92552), anti-Cyclin A1 (ab53699), anti-Cyclin D1 (ab53699), anti-Cyclin E1 (ab133266), and anti-ACTIN antibody (ab8226) that were purchased from Abcam (Cambridge, UK), the next day the PVDF membrane was incubated with a secondary antibody (Beijing TDY Biotech Co, Ltd, Beijing, China) for 1 h. An ECL detection kit (GE Healthcare, UK) was used for blots visualization using and bolts were exposed with X-ray film.
Ab133266
Manufactured by Abcam
Sourced in United Kingdom
Ab133266 is a monoclonal antibody designed for use in various research applications. It recognizes a specific target antigen, but details about its intended use or function are not provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab8226, by Abcam (2 mentions)
Ab62461, by Abcam (1 mentions)
Ab53699, by Abcam (1 mentions)
Ab92552, by Abcam (1 mentions)
Ecl detection kit, by GE Healthcare (1 mentions)
Pdvf membrane, by Merck Group (1 mentions)
Total cell protein extraction kit, by Keygen Biotech (1 mentions)
Ab199728, by Abcam (1 mentions)
Ab124821, by Abcam (1 mentions)
Ab156034, by Abcam (1 mentions)
Ab181602, by Abcam (1 mentions)
Nd 2000, by Thermo Fisher Scientific (1 mentions)
Ffp19, by Beyotime (1 mentions)
Hrp conjugated goat anti rabbit antibody, by Beyotime (1 mentions)
Cyclin e1, by Abcam (1 mentions)
A0208, by Beyotime (1 mentions)
Ab53519, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab92547, by Abcam (1 mentions)
3 protocols using ab133266
1
Total Protein Extraction and Western Blot
2
Western Blot Analysis of Cell Cycle Regulators
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RIPA (Abcam, ab156034, England) is used to lyse cells with a protease inhibitor (Transgen, DI111-01, China) in the ice bath for 30 minutes. Then, the Lysis products were centrifuged at 12000g and the supernatant was transferred to a new tube for protein quantification by a nanodrop (Thermo Scientific, ND-2000, USA). 20μg protein was added to the polyacrylamide gel. After electrophoresis, the protein was transferred to the PVDF membrane overnight at 4°C. The PVDF membrane (Beyotime, FFP19, China) was sealed with 5% skim milk powder for 1 hour and incubated overnight with a primary antibody. Incubate with secondary antibody for 1 h at room temperature the next day. The luminescent solution is then added for development. The list of primary antibodies is as follows: GAPDH (Abcam, ab181602, England), CDK4 (Abcam, ab199728, England), CDK6 (Abcam, ab124821, England), Cyclin D1 (Abcam, ab199728, England), Cyclin E1 (Abcam, ab133266, England). The secondary antibody used an HRP-conjugated goat anti-rabbit antibody (Beyotime, A0208, China).
3
Protein Expression Analysis by Western Blot
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The total protein was extracted from cells with RIPA lysis buffer and the protein concentration was determined using a BCA kit. 20 µg of protein was loaded into the SDS-PAGE gel for electrophoresis, and the protein was then electroporated into the PVDF membrane for blocking and subsequent primary and secondary antibody incubation. Exposure and capture of images were conducted using a chemiluminescent imaging system (Clinx Science Instruments). Primary antibodies were purchased from Abcam and beta-actin was used as an internal reference. The antibodies used in this experiment were: α-SMA (1:1000, ab32575, Abcam); Vimentin (1:1000, ab92547, Abcam); Snail (1:1000, ab53519, Abcam); Cyclin D1 (1:2000, ab16663, Abcam); Cyclin E (1:500, ab133266, Abcam); β-actin (1:2000, ab8226, Abcam).
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