22Rv1, VCaP, and LNCaP cells were lysed in RIPA Lysis and Extraction buffer (Thermo Fisher Scientific), and protein concentration was measured using the BCA protein assay kit (Pierce, Rockford, IL, USA). Fifty or 10 μg of protein was loaded onto 10% SDS-PAGE gels, and then transferred to PVDF membranes (Roche, Basel, Switzerland). After blocking in 5% skimmed milk at room temperature for 1 h, the membranes were incubated with primary antibodies against SIAH1 (1:1,000; Abcam, ab2237), CPSF1 (1:1,000; Abcam, ab81552), AR (1:2,000; Abcam, ab133273), AR-V7 (1:1,000; Abcam, ab198394), and β-actin (1:1,000; Abcam, ab8226) overnight at 4°C, followed by incubation with secondary antibody (goat anti-rabbit IgG H&L [HRP]; 1:1,000; Abcam, ab205718) for 1 h. Immunoblots were visualized using a chemiluminescence detection system (GE Healthcare, Chicago, IL, USA), and luminescent images were scanned into a computer and analyzed using ImageJ software (National Institute of Health, USA).
Ab81552
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab81552 is a primary antibody product from Abcam. It is designed for use in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab2237, by Abcam (2 mentions)
Ab198394, by Abcam (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Ab133273, by Abcam (1 mentions)
Chemiluminescence detection system, by GE Healthcare (1 mentions)
Goat anti rabbit igg h l hrp, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Protein a g bead, by Thermo Fisher Scientific (1 mentions)
Ab126760, by Abcam (1 mentions)
Ab7291, by Abcam (1 mentions)
Ab9116, by Abcam (1 mentions)
Ab46540, by Abcam (1 mentions)
Ab5408, by Abcam (1 mentions)
Horseradish peroxidase conjugates, by Agilent Technologies (1 mentions)
Ab15597, by Abcam (1 mentions)
Ab9110, by Abcam (1 mentions)
4 protocols using ab81552
1
Western Blot Analysis of Prostate Cancer Cells
2
CPSF1 and SIAH1 Protein Interaction
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22Rv1 cells grown in 10-cm dishes at a confluency of 70%–80% were lysed with NP40 buffer. Then, 22Rv1 cell lysates were used for IP with CPSF1 (2 μg/mg; Abcam, ab81552) or SIAH1 (2 μg/mg; Abcam, ab2237) antibody overnight at 4°C. Protein A/G beads (Thermo Fisher Scientific) were added to the IP reactions and left rotating overnight at 4°C. After washing with cold PBS twice, the beads were pelleted by centrifugation at 13,000 rpm at 4°C for 5 min, re-suspended in 10 μL NUPAGE loading buffer, and centrifuged for 2 min at 13,000 rpm before loading onto an SDS gel for western blot analysis.
3
Immunohistochemical Analysis of CPSF1 in HNSCC
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A tissue microarray containing 224 total tissue cores, including 22 cores of non-neoplastic squamous epithelium and 202 cores of head and neck squamous cell carcinoma representing a broad distribution of primary sites was obtained from the Johns Hopkins University Head and Neck Cancer Tissue Bank. Anti-CPSF1 antibody (ab81552) was purchased from Abcam (Cambridge, UK) and used to perform immunohistochemistry on the tissue microarray at a 1:600 dilution. The processed tissue microarray was analyzed for percentage of positively stained epithelial tissue in each sample by two independent reviewers, including a senior pathologist (AAM), and discrepant values were reconciled by consensus. Statistical testing of mean percentage of positively stained epithelial tissue was performed with two-sided, unpaired student’s t-test.
4
Antibody Characterization for hBRG1 and dBRM
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The antibody used to immunoprecipitate hBRG1 was the anti-ratBRG1 rabbit polyclonal antibody raised and characterized by Östlund Farrants et al. (27 (link)). The anti-ratBRG1 was also used for IP of endogenous dBRM in S2 cells. The cross-reactivity of this antibody against dBRM was shown by Tyagi et al. (11 (link)). Western blot analysis of endogenous dBRM was performed using a rabbit antibody raised against the C-terminal part of ct-BRM by Tyagi et al. (11 (link)). The rabbit anti-SNR1 and anti-MOR antibodies were raised and characterized by Dingwall et al. (28 (link)) and Mohrmann et al. (29 (link)), respectively. We also used the following commercial antibodies from Abcam: anti-hBrm (ab15597), anti-RNAPII CTD (ab5408), anti-tubulin (ab7291), anti-hCPSF1 (ab81552), anti-hCPSF2 (ab126760), anti-HA tag (ab9110), anti-V5 tag (ab9116) and anti-IgG (ab46540). Secondary antibodies for Western blotting were horseradish peroxidase conjugates purchased from DakoCytomation.
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