The total protein was extracted from cultured melanoma cells using the RIPA buffer (Beyotime, Shanghai, China) to detect the expression level of CCT3. The protein concentration of each sample was determined using the BCA protein assay kit (Blue Skies, Shanghai, China). 10% SDS-PAGE gel electrophoresis was used for separating total proteins, then the proteins were transferred onto polyvinylidene difluoride membranes. After the membranes were blocked with 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies: mouse anti-GAPDH (1:2000, Santa Cruz, sc-2005), mouse anti-β-actin (1:10000, Abcam,ab6276), rabbit anti-CDK1 (1:300, Abcam, ab32094), rabbit anti-FOXM1 (1:400, Abcam, ab180710), rabbit anti-MCM-2 (1:1000, Abcam, ab109271), rabbit anti-NFKBIA (1:300, Abcam, ab7217), rabbit anti-PIM1 (1:100, Abcam, ab75776), and rabbit anti-SKP2 (1:300, Abcam, ab183039), rabbit anti-Bcl-2 (1:500, Abcam, ab692), rabbit anti-Bax (1:500, Abcam, ab32503), and rabbit anti-Cleaved PARP (1:1000, Abcam, ab32064). The membranes were then washed with TBST 3 times and incubated with the secondary antibody (Yeasen, Shanghai, China) for 2 h at room temperature. Bands were visualized using a chemiluminescent HRP substrate (Millipore, MA, USA) and an electrogenerated chemiluminescence imaging system (Tanon, Shanghai, China) 26 .
Ab32094
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab32094 is a laboratory equipment product. It serves a core function related to scientific research and analysis, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (3 mentions)
Ab181593, by Abcam (3 mentions)
Ab32053, by Abcam (3 mentions)
Ab180710, by Abcam (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Ab6721, by Abcam (2 mentions)
Ab181602, by Abcam (2 mentions)
Ab8227, by Abcam (2 mentions)
Ab692, by Abcam (1 mentions)
Ab183039, by Abcam (1 mentions)
Ab7217, by Abcam (1 mentions)
Ab75776, by Abcam (1 mentions)
Ab32064, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Chemiluminescent hrp substrate, by Merck Group (1 mentions)
Ab6276, by Abcam (1 mentions)
Benzonase nuclease, by Merck Group (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Ab197586, by Abcam (1 mentions)
Ab92552, by Abcam (1 mentions)
12 protocols using ab32094
1
Protein Expression Analysis in Melanoma Cells
2
Quantification of CDK1 and Cyclin B1 in Mouse Embryos
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Recombinant mouse His-GST CDK1 and recombinant mouse 10xHis CCNB1Myc (Creative BioMart) were used as protein standards and loaded at 1, 5, 10 ng per lane.
Wild-type mouse embryo samples containing 130 uninjected embryos and 130 embryos injected with CyclinB1–Venus mRNA at the zygote stage were harvested at the two-cell stage, and embryo extracts were prepared using lysis buffer (150 mM KCl, 20 mM Hepes, pH 7.6, 2 mM EGTA, 1.5 mM MgCl2, 50 mM NaF, 0.1% NP-40, 10% glycerol, 1 mM Na3VO4, 20 mM β-glycerophosphate, 1 mM dithiothreitol, 10 mM benzamidine HCl, and 25 U/ml Benzonase nuclease [Merck]) supplemented with Protease inhibitor cocktail (P2714; Sigma-Aldrich). Protein samples were separated by SDS-PAGE and analyzed by Western blotting, following standard protocols. Transferred nitrocellulose membranes (Immobilon-P Membrane, IPVH00010; Merck) were blotted with anti-CDK1 antibody (YE324, ab32094; Abcam), anti-Cyclin B1 antibody (EPR17060 ; ab181593; Abcam) and subsequently with protein A/G-HRP (recombinant protein A/G, peroxidase conjugated; 32490; Pierce). Scanned images were imported into Adobe Photoshop CS6 and the bands were quantified using ImageJ 1.49d (https://imagej.nih.gov/ij/ ).
Wild-type mouse embryo samples containing 130 uninjected embryos and 130 embryos injected with CyclinB1–Venus mRNA at the zygote stage were harvested at the two-cell stage, and embryo extracts were prepared using lysis buffer (150 mM KCl, 20 mM Hepes, pH 7.6, 2 mM EGTA, 1.5 mM MgCl2, 50 mM NaF, 0.1% NP-40, 10% glycerol, 1 mM Na3VO4, 20 mM β-glycerophosphate, 1 mM dithiothreitol, 10 mM benzamidine HCl, and 25 U/ml Benzonase nuclease [Merck]) supplemented with Protease inhibitor cocktail (P2714; Sigma-Aldrich). Protein samples were separated by SDS-PAGE and analyzed by Western blotting, following standard protocols. Transferred nitrocellulose membranes (Immobilon-P Membrane, IPVH00010; Merck) were blotted with anti-CDK1 antibody (YE324, ab32094; Abcam), anti-Cyclin B1 antibody (
3
Protein Expression Analysis by Western Blotting
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Total proteins were extracted from all cells and BCA method was used to detect protein concentration. The 10% SDS-PAGE was prepared and the gel electrophoresis board was put into the electrophoresis tank. Then, protein samples were subjected to SDS-PAGE electrophoresis. After electrophoresis, the protein samples were transferred to nitrocellulose membrane, which was transferred at the current of 95 mA for 3 h. After incubating in a blocking fluid containing skimmed milk powder for 4 h, membrane was incubated with ANXA7 (ab197586; Abcam; dilution, 1:2000), cell nuclear antigen (PCNA) (ab92552; Abcam; dilution, 1:2000), KI67 (ab16667; Abcam; dilution, 1:1000), cyclin dependent kinase 1 (CDK1) (ab32094; Abcam; dilution, 1:2000), cyclinB1 (ab181593; Abcam; dilution, 1:2000) and CDC5L (ab129114; Abcam; dilution, 1:2000) overnight at 4 °C. The next day, membrane was washed with TBST buffer containing 0.1% Tween20 for four times and incubated with goat anti-rabbit IgG-HRP second antibody (ab6721; Abcam; dilution, 1:2000) at room temperature for 1 h. After the washing of TBS, membrane was disposed with ECL and photographed with a Vilber Lourmat in the darkroom. Image J analysis system was used to analyze the gray values of each band.
4
Protein Expression Analysis Protocol
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Total protein was extracted from transfected cells by RIPA lysis buffer (Beyotime, Shanghai, China). The purity of the protein in the whole extract was determined by bicinchoninic acid (BCA) protein assay kit (Pierce, Appleton, WI). The proteins were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). After incubation with bovine serum albumin (BSA) in Tris-HCl buffered saline containing 0.1% TBST, the membrane was added with corresponding primary antibodies CDK1 (ab32094, 1:2000, Abcam, Cambridge, MA), CCNB1 (ab32053, 1:50000, Abcam, Cambridge, MA), p53 (ab32389, 1:1000, Abcam, Cambridge, MA), p21 (ab109520, 1:1000, Abcam, Cambridge, MA), and GAPDH (ab181602, 1: 10, 000, Abcam, Cambridge, MA). Second antibody Goat anti-rabbit IgG H&L (horseradish peroxidase, HRP) (ab6721, 1:2000, Abcam, Cambridge, MA, United States) was then used to incubate the membrane. The positive bands were detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore) and the strength of the target strip was quantified by Image Lab Software (Bio-Rad).
5
Protein Extraction and Analysis for Tumor Tissues
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Tumor tissues were ground in liquid nitrogen and treated with RIPA for 15∼30 min. Then the tissues or cells were ultrasonicated at 5 s × 4 times and centrifuged at 4°C and 10 000×g for 15 min. The supernatant was stored at −20°C. The protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to PVDF membrane. After block by 5% skim milk for 2 h, the membrane was incubated in primary antibody (1:1000) at 4°C overnight. Next, the membrane was further incubated in secondary antibody (1:1000) at room temperature for 30 min. After washing with phosphate-buffered saline/Tween, the membrane was mixed with a chemiluminescent agent for 1 min and developed. Protein image processing system software and Quantity One software were adopted for scanning and calculation. Antibody: anti-p-VEGFR2 (ab38473, Abcam, USA); anti- VEGFR2 (ab11939, Abcam, USA); anti-PI3K (12402S, Cell Signaling, USA); anti-p-AKT (4060S, Cell Signaling, USA); anti-AKT(9272S, Cell Signaling, USA); anti-Cyclin B1 (ab181593, Abcam, USA); anti-Cyclin A(ab185619, Abcam, USA); anti-CDK1(ab32094, Abcam, USA); anti-Bax (ab32053, Abcam, USA); anti-Bcl-2(ab182858, Abcam, USA); and anti-GADPH(ab9485, Abcam, USA).
6
Immunoblotting of DNA Damage Markers
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Primary antibodies against γH2AX (ab26350, 1:1000), pH3 Ser10 antibody (ab5176, 1:1000), pKAP1 (ab70369, 1000), pCDK Y15 (ab275958, 1:1000), RPA (ab2175, 1:1000), KAP1 (ab22553, 1:1000), CDK1 (ab32094, 1:1000), and FBH1 (ab58881, 1:100) were from Abcam. BrdU antibodies (recognizes IdU) was from BD biosciences (347580, 1:50). pRPA S4/8 antibodies (A300-245A, 1:1000) was from Fortis Life Sciences. Tubulin antibodies was from Novus Biologicals (NB100-690, 1: 1000).
7
Protein Expression Analysis by Western Blot
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Cells were collected in RIPA buffer containing 100 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxysulfonate, and 0.1% SDS, pH 7.4, and protease and phosphatase inhibitors were added to the mixture (Sigma-Aldrich). The protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). The same amount of protein (25 μl) was electrophoresed by 10% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (Hybond, USA), and then probed on the membrane with the following antibodies. Antibodies against SHCBP1 (ab184467), CDK1 (ab32094), and cyclinB (ab32053) were purchased from Abcam (Cambridge, UK). The anti-β-actin was purchased from ABclonal (Wuhan, China). The enhanced chemiluminescence system (ECL kit, KF001, affinity) was used to detect proteins in the membrane.
8
Quantitative Analysis of CDK1 in Liver Tissue
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The liver tissues were taken and washed three times with phosphate buffer solution, then stored at -80 °C. Total proteins were extracted from the liver using RIPA buffer (Beyotime, Nantong, China), and the concentrations were estimated using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL, United States). Next, 30 μg of protein was separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis on a 10% gel and transferred to PVDF membranes. The membranes were blocked in 3% nonfat dry milk and then probed with anti-CDK1 antibody (1:2000 dilution; ab32094; Abcam, Cambridge, MA, United States), and anti-beta actin antibody (1:3000; ab8227; Abcam, Cambridge, MA, United States) overnight at 4 °C. On the next day, membranes were incubated with appropriate secondary antibody for 1 h at room temperature. The protein bands were visualized using Amersham Hyperfilm™ ECL reagent (Thermo Fisher Scientific, MA, United States).
9
Immunoprecipitation and Western Blot Analysis
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Magnetic Beads Protein G were coated with 5 µg of the primary antibody in IP wash buffer (50 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 1 mM MgCl2, and 0.05% NP-40) containing 5% BSA overnight with rotation at 4°C. Then, we collected mouse testes or specific cells and added IP lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 0.5 mM DTT, and 1 mM PMSF/cocktail) followed by incubation on ice for 10 min. Next, we centrifuged the IP lysate at 14,000 rpm for 10 min at 4°C, removed 100 µl of the supernatant and added this to 900 µl of the beads–antibody complex in IP Buffer (860 µl IP wash buffer and 35 µl 0.5 M EDTA) and incubated this with rotation overnight at 4°C. After washing, 50 µl elution buffer was added to the immunoprecipitate and the supernatant was used for Western blot. Western blotting was performed as described previously (Wu et al., 2005 (link)). The commercial antibodies used were anti-CDK1 (Abcam, ab32094) and anti-cyclin B3 (C517).
10
Quantitative Protein Expression Analysis
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Cell samples were lyzed by using RIPA buffer (Beyotime, Shanghai, China). Denatured samples (20 µg per lane) were separated by using 10% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (EMD Millipore, USA). The membranes were incubated with primary antibodies against ACTL8 (1:500, ab96756, Abcam), BIRC5 (1:500, ab469, Abcam), FOXM1 (1:500, ab180710, Abcam), CDK1 (1:1,000, ab32094, Abcam), STMN1 (1:1,000, #9562, CST), CCNB1 (1:1,000, #4138, CST), PLK1 (1:1,000, #4513, CST), STMN1 (1:1,000, #9562, CST) and GAPDH (1:4,000, sc-32233, Santa-Cruz). After washing, secondary antibodies that conjugated to HRP were added and incubated for color development. Densitometry analyses were performed using Quantity One software (Bio-Rad Laboratories, Mississauga, Canada).
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