For co-immunoprecipitation, HEK293 cells in 10-cm culture plates were transiently transfected with 6 μg of the indicated plasmids, cultured for 48 hours after transfection, and lysed in lysis buffer (150 mM NaCl, 20 mM Tris-HCl, 1.5 mM MgCl2, 1% NP-40, 15% glycerol, 2 mM EDTA) with protease inhibitor cocktails (P8345-5ML, Sigma-Aldrich) at a dilution of 1:100. After precleaning cell lysates with protein A/G plus-agarose beads (sc-2003, Santa Cruz) for 1 hour and blocking anti-Flag (A2220-1ML, Sigma-Aldrich)/anti-GFP antibody-conjugated agarose beads with 2.5% albumin/bovine (94349-60-7, Acros Organics, Thermo Fisher, Waltham, MA) for 1 hour, cell lysates were added to anti-FLAG/anti-GFP antibody-conjugated agarose beads and rotated at 4° C for 1.5 hours. The beads were washed using lysis buffer four times. Immunoprecipitates and total cell lysates were boiled in SDS loading buffer for 10 minutes and then subjected to Western blot analysis using anti-Flag antibody or anti-GFP antibody.
P8345 5ml
Manufactured by Merck Group
P8345-5ML is a lab equipment product from Merck Group. It is a 5 mL container used for various laboratory applications. The product's core function is to provide a contained storage solution for liquids and powders in a laboratory setting.
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Lab products found in correlation
2 protocols using p8345 5ml
1
Co-Immunoprecipitation of Flag- and GFP-Tagged Proteins
2
Co-Immunoprecipitation Assay for Protein Interactions
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For co-immunoprecipitation, HEK 293 cells in 10-cm culture plates were transiently transfected with 6 μg of the indicated plasmids, cultured for 48 hours after transfection, and lysed in lysis buffer (150 mM NaCl, 20 mM Tris-HCl, 1.5 mM MgCl2, 1% NP-40, 15% glycerol, 2 mM EDTA) with protease inhibitor cocktails (P8345-5ML, Sigma) at a dilution of 1:100. After precleaning cell lysates with protein A/G plus-agarose beads (sc-2003, Santa Cruz) for 1 hour and blocking anti-Flag (A2220-1ML, Sigma)/anti-MYC antibody-conjugated agarose beads (A7470-1ML, Sigma) with 2.5% albumin/bovine (94349-60-7, Acros organics) for 1 hour, cell lysates were added to anti-FLAG/anti-MYC antibody-conjugated agarose beads and rotated at 4°C for 1.5 hours. The beads were washed using lysis buffer four times. Immunoprecipitates and total cell lysates were boiled in SDS loading buffer for 10 minutes and then subjected to Western blot analysis using anti-Flag antibody (F1804-200UG, Sigma) and anti-MYC antibody (06-549, Sigma).
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