Total protein from OC tissues and cells was extracted using protein extraction lysate (Beyotime, Shanghai, China). Then, protein concentration was evaluated by BCA Protein Assay Kit (Beyotime, Shanghai, China). Protein samples were separated by 10% SDS-PAGE and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). Next, PVDF membranes were blocked with 5% nonfat milk for 2 h and incubated overnight at 4°C with primary antibody against CDCA7 (Thermo Fisher Scientific, PA5-101,299, 1:1000), EZH2 (Thermo Fisher Scientific, MA5-15,101, 1:1000), CyclinE1 (Abcam, ab33911, 1:1000), CyclinE2 (Abcam, ab40890, 1:5000), MMP2 (Abcam, ab97779, 1:2000), MMP9 (Abcam, ab228402, 1:1000), VEGFA (Abcam, ab214424, 1:1000), VEGFR1 (Abcam, ab32152, 1:5000), VEGFR2 (Abcam, ab134191, 1:5000) and GAPDH (Thermo Fisher Scientific, MA5-15,738-D680, 1:1000). The next day, membranes were washed with TBST and incubated with the corresponding secondary antibody (Thermo Fisher Scientific, A32728, 1:10,000) for 2 h at room temperature. Enhanced chemiluminescence reagents (ECL; Pierce, IL, USA) were applied to visualize the protein bars. Finally, protein bands were detected by a Bio-Rad imaging system (Bio-Rad, CA, USA).
Ab33911
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Ab33911 is a lab equipment product offered by Abcam. It is a device used for scientific research purposes. The core function of this product is to assist with laboratory procedures, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab134175, by Abcam (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ab32147, by Abcam (5 mentions)
Ab16663, by Abcam (5 mentions)
Ab181602, by Abcam (5 mentions)
Ab181591, by Abcam (4 mentions)
Ab32124, by Abcam (4 mentions)
Ab108357, by Abcam (4 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Ab8245, by Abcam (3 mentions)
Ab32503, by Abcam (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Ab40890, by Abcam (2 mentions)
Imaging system, by Bio-Rad (2 mentions)
Ab32042, by Abcam (2 mentions)
Nitrocellulose membrane, by LI COR (2 mentions)
Protein assay dye, by Bio-Rad (2 mentions)
Mouse anti α tubulin t5168, by Merck Group (2 mentions)
Anti rabbit irdye 800, by LI COR (2 mentions)
36 protocols using ab33911
1
Protein Expression Analysis in Ovarian Cancer
2
Phillyrin Modulates Oxidative Stress and Apoptosis in ARPE-19 Cells
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Human ARPE-19 cell line was purchased from Beijing Beina Chuanglian Biotechnology Research Institute (Beijing, China). Phillyrin (batch number Z17A8X34077, purity > 98%) was a product of the China Food and Drug Administration (Beijing, China). Dulbecco's Modified Eagle's Medium/nutrient mixture F-12 (DMEM/F-12), fetal bovine serum (FBS), penicillin, and streptomycin solutions were purchased from Corning (NY, USA). Trypsin (0.05%) and phosphate buffered saline (PBS) were produced by Gibco, Invitrogen (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT), ML385, N-acetylcysteine (NAC)m and H2O2 solution were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against Fas (ab82419), cytochrome c (ab90529), cleaved caspase-3 (ab2302), cleaved caspase-9 (ab2324), NQO1 (ab80588), Keap1 (ab118285), Bcl-2 (ab185002), Nrf2 (ab62352), CDK2 (ab32147), cyclin A (ab33911), cyclin E (ab181591), Bax (ab53154), β-actin (ab8226), p21 (ab188224), Histone H3 (ab1791), COX IV (ab16056) p53 (ab241556), pro caspase-3, pro caspase-8, and pro caspase-9 were obtained from Abcam, Cambridge, (MA, USA). Antibodies against p-p53 (9286S), cleaved caspase-8 (9496S), cleaved PARP (5625S), and HO-1 (86806S) were purchased from Cell Signaling Technology, Beverly, (MA, USA).
3
Western Blot Analysis of Cellular Proteins
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Cells were lysed with RIPA lysis buffer (Beyotime, Haimen, China) supplemented with a protease inhibitor cocktail (Beyo-time). Protein concentrations were determined by using a BCA protein assay kit (Beyotime). Cell lysates were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The antibodies used in this assay were: rabbit polyclonal anti-human SIRT4 antibody (clone HPA029691; Sigma), rabbit monoclonal anti-human cyclin D antibody (60816-1-IG; Proteintech, Rosemont, IL, USA), rabbit monoclonal anti-human cyclin E antibody (Ab33911; Abcam, Lon-don, UK), rabbit polyclonal anti-human ERK antibody (9102; CST, Danvers, MA, USA), rabbit polyclonal anti-human p-ERK antibody (4370; CST), rabbit polyclonal anti-human caspase 3 (35/18 kD) antibody (9662; CST), rabbit polyclonal anti-human caspase 9 (46 kD) antibody (10380-1-AP; Pro-teintech), rabbit polyclonal anti-human p65 (65 kD) antibody (10745-1-AP; Proteintech), rabbit polyclonal anti-human MMP9 (78 kD) antibody (10375-2-AP; Proteintech), rabbit monoclonal anti-human n-cadherin (99 kD) antibody (14472; CST), rabbit monoclonal anti-human e-cadherin (135 kD) antibody (Ab124397; Abcam, Cambridge, UK), goat anti-rabbit detection antibody (ab97200; Abcam), and rabbit polyclonal anti-human GAPDH antibody (AB-P-R 001; Hangzhou Good-here Biotechnology Co., Ltd., Hangzhou, China).
4
Protein Expression Analysis in HRMECs
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HRMECs were collected and extracted by RIPA lysis buffer (89901, Thermo Scientific, U.S.A.) with protease inhibitor cocktail (Merck KGaA, Darmstadt, Germany). A BCA™ Protein Assay Kit (Pierce, Appleton, WI, U.S.A.) was used to evaluate the protein levels. Equal amounts of proteins were loaded on 10% SDS-PAGE gels (Thermo Scientific), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, Massachusetts, U.S.A.) and blocked with 5% non-fat milk at room temperature for 1 h. Afterwards, membranes were subjected to immunoblotting with primary antibodies (all purchased from Abcam, Cambridge, MA, U.S.A.) against SOCS6 (ab197335, 1/500), p-JAK2 (ab195055, 1/500) JAK2 (ab39636, 1/1000), p-STAT3 (ab32143, 1/1000) STAT3 (ab119352, 1/5000), PCNA (ab92552, 1/1000), Cyclin D1 (ab16663, 1/200), Cyclin E1 (ab33911, 1/1000), cleaved caspase-3 (ab32042, 1/500), Bcl-2 (ab59348, 1/500) and Bax (ab216494, 1/100) and incubated at 4°C overnight. Subsequently, the blots were washed with PBST, and the membranes were incubated with secondary peroxidase-linked goat anti-rabbit IgG H&L (1:5000, Santa Cruz, California, U.S.A.) at room temperature for 2 h. Signals were visualized by the ECL (Amersham Biosciences, Piscataway, NJ) method after washing, while optical densities of the bands were measured using ImageJ software (Bio-Rad).
5
Immunoblotting analysis of cellular proteins
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Anti-COX2/Cyclooxygenase 2 antibody (ab179800), anti-Cleaved PARP1 antibody (ab32561), anti-PARP antibody (ab74290), anti-XIAP antibody (ab21278), anti-MMP9 antibody (ab73734), anti-Cyclin E1 antibody (ab33911) and anti-GAPDH antibody (ab181602) were purchased from Abcam (Cambridge, U.K.) and were used as primary antibodies. Proteins were collected on ice using NP40 cell lysis buffer for 30 min and then were separated by 8% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). Then the gel was transferred on to polyvinylidene difluoride (PVDF) membrane for 2 h. Five percent milk-TBST (TBS with 0.05% Tween 20) was used for 1 h to block membrane and then was incubated with specific primary antibodies overnight at 4°C. Before incubated by secondary antibody for 1.5 h, the membrane was washed by TBST. ECL reagent was used to visualize proteins and ImageJ software was used to analyze the obtained data.
6
Western Blot Analysis of Cell Signaling Proteins
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After 0 μM, 0.5 μM, 1.0 μM, or 1.5 µM 7f treatment of A549 or PC-9 cells, performed western blot analysis according to relevant general operating procedures. Primary antibodies (100 μL/cm2) included those against c-myc (ab185656, Abcam Company, USA), cyclin D (ab16663, Abcam Company, USA), cyclin E (ab33911, Abcam Company, USA), Ki67 (ab16667, Abcam Company), P53 (SC-126, Santa Cruz Company, USA), MDM2 (ab38618, Abcam Company, USA), cleaved caspase-3 (YT6161, Immunoway Company, USA), caspase-3 (YT6113, Immunoway Company, USA), bax (50599–2-lg, Proteintech Company, USA), VEGFR-2 (#9698, Cell Signaling Technology, USA), β-actin (60012–1-lg, Proteintech Company, USA), bcl-2 (12789–1-AP, Proteintech Company, USA), VDCA-1 (ab154856, Abcam Company, USA), p-VEGFR-2 (#3770, Cell Signaling Technology, USA), and Cytochrome C (ab133504, Abcam Company, USA).
7
Protein Extraction and Western Blot Analysis
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Proteins were collected from cells using the Whole Cell Lysis Assay Kit (KGP250, KeyGen, Nanjing, China). The protein concentration was determined using the bicinchoninic acid (BCA) method using the BCA Protein Quantitation Assay Kit (KeyGen, Nanjing, China). Protein was electrophoretically separated by 10% or 15% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA). The membranes were blocked for 1 hour with 5% bovine serum albumin (BSA) in TBS-T and incubated with specific primary antibodies overnight at 4° C followed by incubation with rabbit or mouse radish peroxidase-coupled secondary antibodies for 1 hour. Antibody binding was detected using the enhanced chemiluminescence reagent (Millipore, Billerica, MA). The antibodies used in this study were as follows: ALKBH5 (ab195377, Abcam), H3K27ac (#8173, Cell Signaling Technology [CST]), cyclin D1 (#2978, CST), cyclin E1 (ab33911, Abcam), c-Myc (#5605, CST), caspase-3 (19677-1-AP, Proteintech), BCL-2 (12789-1-AP, Proteintech), MMP2 (#87809, CST), MMP7 (#71031, CST), MMP9 (#13667, CST), E-cadherin (#3195, CST), N-cadherin (#13116, CST), vimentin (#5741, CST), β-catenin (#8480, CST), Snail (ab53519, Abcam), Slug (ab27568, Abcam), β-actin (ab8227, Abcam), and GAPDH (#8884, CST).
8
Characterization of MCM Protein Expression
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For WB, the detailed procedures were performed as described previously [8] using the following primary antibodies: anti-MCM2 (Abcam, ab4461), anti-MCM3 (Abcam, ab128923), anti-MCM4 (Abcam, ab4459), anti-MCM5 (Abcam, ab75975), anti-MCM6 (Abcam, ab201683), anti-MCM7 (Abcam, ab2360), anti-CDK9 (Abcam, ab76320), anti-CyclinD1 (Abcam, ab16663), anti-CyclinE1 (Abcam, ab33911), anti-p53 (Abcam, ab241566), anti-p21 (Abcam, ab109502), anti-p27 (Abcam, ab32034), and anti-GAPDH (Abcam, ab181602).
For IHC, the standard method was described previously.14 (link) Slides were incubated with primary antibodies (Abs) against MCM2 (Immuway, YM6642), MCM3 (Abcam, ab128923), MCM4 (Immuway, YT2681), MCM5 (Abcam, ab75975), MCM6 (Abcam, ab190948), and MCM7 (Abcam, ab2360).
For IHC, the standard method was described previously.14 (link) Slides were incubated with primary antibodies (Abs) against MCM2 (Immuway, YM6642), MCM3 (Abcam, ab128923), MCM4 (Immuway, YT2681), MCM5 (Abcam, ab75975), MCM6 (Abcam, ab190948), and MCM7 (Abcam, ab2360).
9
Breast Cancer miRNA and Protein Expression
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The total RNA was extracted with RNAiso Plus (Takara, Kusatsu, Japan) from collected clinical samples and chosen breast cancer cells, according to the manufacturer's protocol. cDNA was specifically synthesized for miRNA, and then was tested using the Hairpin-it miRNA qPCR Quantitation Kit (GenePharma, China). Other cDNA was synthesized by the reverse transcription kit (Takara), and then detected by the SYBR Green Master Mix Kit (Roche, Reinach, Switzerland). The expressions were normalized based on U6 or GAPDH, respectively. The primers for targets are listed in Supplementary Table 1 .
The primary antibodies utilized for western blot were as follows: anti-CCNE1 (Abcam, Cambridge, UK, 1:1,000, ab33911), anti-CDK2 (Abcam, 1:3,000, ab32174), anti-c-Myc (Abcam, 1:1,000, ab32072), anti-E2F1 (Abcam, 1:1,000, ab 112580), and anti-GAPDH (Abcam, 1:2,500, ab9485). The secondary antibodies (GOAT anti-mouse and anti-rabbit IgG) were from Jackson Immunoresearch (USA).
The primary antibodies utilized for western blot were as follows: anti-CCNE1 (Abcam, Cambridge, UK, 1:1,000, ab33911), anti-CDK2 (Abcam, 1:3,000, ab32174), anti-c-Myc (Abcam, 1:1,000, ab32072), anti-E2F1 (Abcam, 1:1,000, ab 112580), and anti-GAPDH (Abcam, 1:2,500, ab9485). The secondary antibodies (GOAT anti-mouse and anti-rabbit IgG) were from Jackson Immunoresearch (USA).
10
Protein Quantification and Western Blot Analysis
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Cells were lysed in RIPA buffer containing Halt protease inhibitor cocktail (Pierce, Rockford, IL, USA) and phosphatase inhibitors (Cayman Chemical, Ann Arbor, MI, USA), and the protein concentration was determined by the Bradford method. Sodium dodecyl sulfate-polyacrylamide agarose gel electrophoresis (SDS-PAGE) was run at 120 V for protein separation. Thereafter, the proteins were transferred from the gel to polyvinylidene fluoride (PVDF) membranes and blocked in TBS-Tween (TBS-T) supplemented with 0.05 g/mL bovine serum albumin for 1 h. The membranes were then incubated with primary antibodies against GAPDH (1:10,000, ab181602), TRIM36 (1:1,000, ab272672), and cyclin E (1:1,000, ab33911) (Abcam, Cambridge, MA, USA) overnight at 4°C. After washing with TBS-T, the membranes were incubated with goat anti-rabbit IgG (1:5,000, Beijing ComWin Biotech Co., Ltd., Beijing, China) for 2 h at room temperature, followed by washing three times in TBS-T. Protein bands were quantified by chemiluminescence imaging analysis system (GE Healthcare, Beijing, China) using an electrogenerated chemiluminescence (ECL) reagent.
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