Cell pellets were resuspended in sample buffer, boiled and analyzed by western blotting. Membranes were probed with the following antibodies: anti-GFP (rabbit polyclonal, Abcam, ab190584, 1:10000), anti-Bub1 (rabbit polyclonal; Abcam, Cambridge, UK; 1:5000), anti-BubR1 (mouse monoclonal; BD #612503, 1:1000) and anti-Tubulin (mouse monoclonal; Sigma; 1:8000), anti-Incenp (rabbit polyclonal, Cell Signaling #2807, 1:500), anti-Cyclin B (mouse monoclonal, Santa Cruz, sc-245, 1:1000), anti-PLK1 (mouse monoclonal; Abcam #ab17057, 1:1000), anti-Vinculin (mouse monoclonal, Sigma, V9131, 1:10000).
Anti bub1
Manufactured by Abcam
Sourced in United Kingdom, United States
Anti-Bub1 is a primary antibody that specifically recognizes the Bub1 protein. Bub1 is a serine/threonine-protein kinase that plays a crucial role in the spindle assembly checkpoint, ensuring proper chromosome segregation during cell division.
Automatically generated - may contain errors
Lab products found in correlation
Anti vinculin, by Merck Group (2 mentions)
Anti mad2, by Fortis Life Sciences (2 mentions)
Anti tubulin, by Merck Group (1 mentions)
Anti incenp, by Cell Signaling Technology (1 mentions)
Anti cyclin b, by Santa Cruz Biotechnology (1 mentions)
Anti plk1, by Abcam (1 mentions)
Anti bubr1, by BD (1 mentions)
Anti gfp, by Abcam (1 mentions)
Ab190584, by Abcam (1 mentions)
Ab17057, by Abcam (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti p62, by Santa Cruz Biotechnology (1 mentions)
Anti histone h3, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Anti cathepsin d, by Abcam (1 mentions)
Anti lc3, by Merck Group (1 mentions)
Anti gfp, by Roche (1 mentions)
6 protocols using anti bub1
1
Western Blot Analysis of Mitotic Proteins
2
Comprehensive Protein Analysis Protocol
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For protein analyses, cells were lysed in lysis buffer (50 mM Tris-HCl at pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 0.5% NP-40, 10% glycerol, protease inhibitor cocktail [Roche], phosphatase inhibitor cocktail [Roche]) and resolved on 15% SDS-PAGE gels. The following primary antibodies were used: anti-LC3 (1:1000; Sigma-Aldrich), anti-Flag (1:1000; Sigma-Aldrich), anti-actin (1:10,000; Sigma-Aldrich), anti-GAPDH (1:2000; Cell Signaling), anti-Mad2 (1:1000; Bethyl Laboratories), anti-Bub1 (1:2000; Abcam), anti-vinculin (1:5000; Sigma-Aldrich), anti-cathepsin D (1:2000; Abcam), anti-GFP (1:5000; Roche), anti-Histone H3 (1:1000; Cell Signaling), and anti-p62 (1:500; Santa Cruz Biotechnology).
3
Kinetochore Protein Staining Protocol
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For staining of kinetochore proteins, cells on coverslips were pre-extracted with PHEM-buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgSO4) supplemented with 0.5 % Triton-X-100 for 15 min and after that fixed with 2 % paraformaldehyde in 0.5 % Triton-X-100/PHEM for 15 min. For the whole cell analysis, cells were fixed directly with 2 % paraformaldehyde in 0.5 % Triton-X-100/PHEM for 15 min and then rinsed with MBST (10 mM MOPS, 150 mM NaCl and 0.05% Tween 20). Next the cells were blocked with 20 % boiled normal goat serum (bngs) in MBST for 1h at RT, followed by staining with primary antibodies for overnight at 4°C or 1h at RT. Primary antibodies used were mouse anti-Mad2 (Santa Cruz, Dallas, TX, USA, sc-65492, 1:75), anti-Bub1 (Abcam, ab9000, 1:150) and human autoimmune serum (Crest, Antibodies Incorporated, Davis, USA, 1:200). The secondary antibody dilutions and sample mounting were performed as described elsewhere [51 (link)].
4
Western Blot Analysis of Cell Cycle Proteins
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We separated equal amounts of protein with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred the protein to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Non-specific protein interactions were blocked with incubation in 3% non-fat milk and Tris-buffered saline with Tween-20 (TBST) at 37 °C for 30 min. Subsequently, the membranes were incubated for 2 h at room temperature with antibodies against CCNA2 (1:1000, Abcam, Cambridge, UK), anti-BUB1 (1:1000, Abcam, Cambridge, UK), anti-CDK1 (Abcam, Cambridge, UK), and anti-GAPDH (Abcam, Cambridge, UK). Following antibody incubations, membranes were incubated with an HRP-conjugated secondary antibody. GAPDH was used as the endogenous control. The target protein bands were visualized. We applied an enhanced chemiluminescence (ECL) reagent kit (Millipore) to visualize target protein bands and exposed the membrane to an X-ray film (Fuji).
5
Western Blot Analysis of Cell Signaling Proteins
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Radioimmunoprecipitation assay buffer supplemented with phenyl methane sulfonyl uoride was used to lyse tumour cells. After the lysate was centrifuged, the supernatant was collected for further analysis. After the protein concentration was measured, equal amounts of protein were loaded onto 10% SDS-PAGE gels and transferred onto polyvinylidene uoride membranes. Then, the membranes were blocked using 5% skimmed milk at 37 ºC for 2 h and were incubated with the primary antibodies (anti-SMAD2/SMAD3, anti-BUB1, anti-PSMAD2/PSMAD3, 1:1,000, Abcam; anti-PDL1, 1:1,000, eBioscience, California; anti-GAPDH, 1:1,000, Proteintech, Chicago) at 4 ºC overnight. Then, the anti-mouse/rabbit antibodies (1:3,000, Beyotime, Shanghai) were used to incubate the membranes at 37 ºC for 1h. The protein bands were detected by the chemiluminescence kit (Thermo Fisher, Shanghai).
6
Western Blot Analysis of Cell Signaling Proteins
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HeLa cells from two wells of a 6-well dish were scraped and spun down at 1,500 rpm to generate cell lysates for Western blotting. Pellets were washed with Dulbecco’s PBS (Invitrogen) and resuspended in 2× SDS sample buffer, sonicated, and run on SDS-polyacrylamide gel. Antibodies used were as follows: anti-survivin (Cell Signaling Technology), anti-tubulin DM1-α (Sigma-Aldrich), anti-AIM1 (BD), anti-EB1 (BD), anti-Mad2 (Bethyl Laboratories, Inc.), and anti-Bub1 (Abcam).
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