Freshly isolated colons were flushed with phosphate-buffered saline (PBS), cut longitudinally and scraped to isolate colonic mucosa. Tissues were homogenized and sonicated in lysis buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.5% Na deoxycholate) supplemented with protease inhibitors. After centrifugation at 35,000 g for 15 min, 10 μg of the supernatants were separated on 4–15% gradient SDS-polyacrylamide gels (Bio-Rad) and transferred onto nitrocellulose membranes (Bio-Rad) and probed at 4°C overnight with the following mouse monoclonal primary antibodies: IQGAP1 (BD Biosciences) at 1:1000 dilution; IQGAP2 (sc-55525, Santa Cruz) at 1:1000; β-actin and α-tubulin (Sigma Aldrich) at 1:2000. Blots were washed and probed with anti-mouse HRP-conjugated secondary antibodies at 37°C for 1 hour. Chemiluminescent detection was used to visualize protein bands.
Iqgap1
Manufactured by BD
Sourced in France, Canada
IQGAP1 is a cytoplasmic protein that functions as a scaffold for various signaling pathways. It interacts with multiple proteins and is involved in regulating cellular processes such as cell-cell adhesion, cell migration, and cell proliferation. The core function of IQGAP1 is to facilitate the coordination and integration of these cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
α tubulin, by Merck Group (2 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
β actin, by Merck Group (1 mentions)
Gradient sds polyacrylamide gel, by Bio-Rad (1 mentions)
Hax 1, by BD (1 mentions)
Goat anti rabbit igg, by Beyotime (1 mentions)
Goat anti mouse igg, by Beyotime (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
3 protocols using iqgap1
1
Colonic Mucosa Protein Extraction and Western Blot
2
Western Blotting of Cytoskeletal Proteins
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Cells were collected in lysis buffer as described (Zaoui et al., 2008 (link)). Western blotting was performed using antibodies directed against mDia1, IQGAP1, HAX1 (BD Biosciences, Le Pont de Claix, France), Dia2c (Santa Cruz Biotechnology, Dallas, TX), mDia2/DRF3 (a kind gift from H. N. Higgs, Dartmouth Medical School, Hanover, NH), α-tubulin, ELKS (Sigma-Aldrich, Lyon, France), and GFP (Roche, Meylan, France).
3
Western Blot Analysis of IQGAP1 and GAPDH
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Cells were washed with PBS twice, dissolved, and vortexed in lysis buffer. The cell extracts were incubated on ice for 30 minutes, centrifuged at 14,000 rpm for 30 minutes, and the supernatants were collected. Protein were loaded and separated on 8% SDS-PAGE gels and transferred onto 0.45-μm transfer membranes (Corning, NY). The membranes were blocked with PBS containing 5% dried milk and 0.05% Tween 20 for 1 hour, and then probed with antibodies against GAPDH (1:2000, Santa Cruz, CA), IQGAP1 (1:5000, BD biosciences, New Jersey). Subsequently, visualization used enhanced chemiluminescence detection kit for HRP (BioInd, Israel) with the appropriate secondary antibodies (goat anti-mouse IgG, 1:1000; goat anti-rabbit IgG, 1:1000; Beyotime, Shanghai, China), which contained horseradish peroxidase.
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