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.
Ab53699
Manufactured by Abcam
Sourced in United Kingdom
Ab53699 is a product offered by Abcam. It is a piece of lab equipment. No further details are available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (3 mentions)
Ab181602, by Abcam (2 mentions)
Ab32503, by Abcam (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ab32147, by Abcam (2 mentions)
Ab40754, by Abcam (1 mentions)
Ab202026, by Abcam (1 mentions)
Ab251892, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab62461, by Abcam (1 mentions)
Ab92552, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab133266, 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)
Ab32174, by Abcam (1 mentions)
Ab57653, by Abcam (1 mentions)
Beyoecl moon, by Beyotime (1 mentions)
Ab133327, by Abcam (1 mentions)
7 protocols using ab53699
1
Total Protein Extraction and Western Blot
2
Western Blot Analysis of Cell Cycle and Apoptosis Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were obtained using RIPA lysis buffer (Beyotime, Shanghai). Afterwards, 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate the proteins. The protein was then transferred onto polyvinylidene fluoride membranes (Millipore). After blocking with 5% skim milk, the membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies against cyclin A1 (ab53699), cyclin B1 (ab251892), cyclin D1 (ab40754), Bcl-2 (ab141523), Bax (ab32503), TP53 (ab202026), and GAPDH (ab181602) were purchased from Abcam Company (UK). Next, the membranes were incubated with the appropriate secondary antibodies for 2 h at room temperature. Pharmacia enhanced chemiluminescence was used to visualize the protein bands. GAPDH functioned as a loading control.
3
Western Blot Analysis of Cell Cycle Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected and lysed with RIPA Lysis Buffer (Beyotime Biotechnology, Beijing, People’s Republic of China). Protein samples were separated on 8% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and transferred to polyvinylidenedifluoride membrane (Millipore, Billerica, USA) for 3 hrs with transfer buffer (25 mM Tris, 192 mM glycine and 20% [v/v] methanol). The membranes were blocked in 5% non-fat dry milk in TBS with 0.1% Tween 20 for 1 hr at room temperature and incubated with primary antibodies at 4°C overnight, including RRM2 (1:1000, ab57653, Abcam), Cyclin A1 (1:1000, ab53699, Abcam), Cyclin B1 (1:1000, ab72, Abcam), CDK2 (1:1000, ab32174, Abcam), p-AKT (1:2000, 4060, CST), total-AKT (1:2000, 4691, CST), GAPDH (1:2000, 2118S, CST), followed by incubation with an HRP-conjugated anti-mouse or anti-rabbit secondary antibody separately. Finally, the bands were imaged by Tanon 5200 using the BeyoECL Moon (Beyotime Biotechnology, Beijing, People’s Republic of China).
4
Western Blot Analysis of Cell Cycle Regulators
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were washed twice with 4°C PBS and then lysed in cold RIPA buffer with protease inhibitors. The BCA Protein Quantitation Assay (KeyGen Biotech, Nanjing, China) was used to measure the protein concentration. The total protein was transferred to a nitrocellulose membrane after denaturing by 10% SDS‐PAGE. The membranes were blocked with 5% non‐fat milk in Tris‐buffered saline containing 0.1% Tween‐20 (TBST) for 1 h at room temperature. The membranes were then incubated with the following primary antibodies overnight at 4°C (dilution ratio 1:2000). The membranes were washed three times with TBST and then incubated with secondary antibodies (anti‐rabbit IgG or anti‐mouse IgG) for 1 h at room temperature. The membranes were washed three times with TBST, and then, the targeted proteins were detected by the ECL (EMD Millipore, MA, USA) method. XPOT, CDK1, CDK2, CDK4, CCNA1, CCNB1, CCNB2, CCNE2 antibodies for western blot were obtained from Bioss (bs‐14673R), Abcam (ab133327), Abcam (ab32147), Abcam (ab108357), Abcam (ab53699), Abcam (ab32053), Abcam (18250), Abcam (40890).
5
Western Blot Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted using RIPA lysis buffer. Equal amounts of proteins were separated by 12% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After being blocked, the membranes were incubated with primary antibodies against COX20 (1:1000, ab18197, Abcam), caspase 3 (1:1000, ab32147, Abcam), cleaved caspase 3 (1:1000, ab53699, Abcam), Bax (1:500, ab32503, Abcam), Bcl-2 (1:500, ab32124, Abcam) and GAPDH (1:1000, ab181602, Abcam) overnight at 4 °C. Protein signals were observed by a chemiluminescence detection system, and relative expression levels of target proteins were analyzed using Image-Pro Plus 6.0.
6
Comprehensive Western Blot Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed as previously described (13 (link)). The antibodies used in the analysis were anti-CPNE1 (1:500; Z6; Santa Cruz Biotechnology, Inc., Dallas, TX, USA); anti-phosphorylated (p)-protein kinase B (AKT; Ser473; 1:1,000 D9E), anti-AKT (1:1,000; 9272s; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-extracellular signal-regulated kinase (ERK; 1:1,000; 137F5), anti-p-ERK (Thr202/Tyr202; 1:1,000; D13.14.4E), anti-matrix metalloproteinase MMP2 (1:1,000; D8N9Y), anti-MMP9 (1:1,000; 603H), anti-Snail (1:1,000; C15D3; Cell Signaling Technology, Inc.); anti-cyclin A1 (1:300; ab53699; Abcam), anti-cyclin B1 (1:300; Ab-147; Abcam), anti-cyclin E1 (1:300; ab33911, Abcam) and anti-GAPDH (1:5,000; D4C6R; Cell Signaling Technology, Inc.) primary antibodies, and peroxidase conjugated anti-mouse or anti-rabbit secondary antibodies (1:5,000; 14709 and 14708; Cell Signaling Technology, Inc.).
7
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues or cells were harvested and lysed with RIPA Lysis Buffer (Beyotime Biotechnology, Beijing, China). The protein lysates were quantified and then separated by 8% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Then, the proteins were transferred to polyvinylidenedifluoride membrane (Millipore, Billerica, USA). The membranes were blocked in 5% non-fat dry milk and incubated with primary antibodies at 4°C overnight, including Cyclin A1 (1:1000, ab53699, Abcam), Cyclin B1 (1:1000, ab72, Abcam), GAPDH (1:2000, 2118S, CST). Then, HRP-conjugated secondary antibodies were incubated for 1 h at room temperature. Finally, the bands were visualized by Tanon 5200.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!