Briefly, 10 × 106 cells were collected and lysed in 1 ml of RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) for immunoprecipitation. Cell extracts were pre-cleared by mixing lysates with 2 μg of normal control IgG and 50 μl of protein G agarose and incubating for 4 h at 4 °C. The pre-cleared lysates were mixed with 2 μg of the appropriate antibodies plus 20 μl of protein G agarose beads and rotated overnight at 4 °C. The immunoprecipitated complexes were obtained by centrifugation, washed three times with RIPA lysis buffer and boiled at 75 °C in loading buffer for 10 min for western blot. Cell lysates were also directly used for western blot analysis. All western blot photos were representative photos from at least two experiments with similar results. Antibodies for HSC 70 (cat no. SC-7298), Lyn (cat no. SC-7274), Fyn (cat no. SC-16), PSTPIP2 (cat no. SC-83015), GATA1 (cat no. SC-1234), SHC (cat no. SC-1695) were purchased from Santa Cruz Biotechnology. Antibody for FLAG (cat no. F3165) was obtained from Sigma. Antibodies for ERK (cat no. 4695), p-ERK (cat no. 4370), p-SRC (cat no. 2101) were purchased from Cell Signaling Technology.
Antibody for flag
Manufactured by Merck Group
Sourced in United States
The Antibody for FLAG is a laboratory reagent used to detect and purify proteins that have been engineered to contain a specific peptide tag, known as the FLAG tag. The antibody binds to this tag, allowing for the identification and isolation of the tagged protein from a sample. The core function of this product is to facilitate the detection and purification of FLAG-tagged proteins in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Gata1, by Santa Cruz Biotechnology (1 mentions)
Antibodies for erk, by Cell Signaling Technology (1 mentions)
Total akt, by Cell Signaling Technology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Mg132, by AG Scientific (1 mentions)
Actinomycin d, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
4 protocols using antibody for flag
1
Immunoprecipitation and Western Blot Protocol
2
Antibody Validation for Cell Signaling
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Antibody for GPNMB (#AF2330) was obtained from R&D systems. Antibody for CD44 (#553131) was obtained from BD Pharmingen. Antibody for NFκB p65 (#39370) was obtained from Active Motif. Antibodies for F4/80 (#ab6640) and Alexa Flour 488-labeled CD11c (#ab33503) were purchased from Abcam. Antibodies for phospho-NFκB p65 (#3033), phospho-Akt (#9271), total-Akt (#9272), and GAPDH (#2118) were purchased from Cell Signaling Technology. Antibody for FLAG (#F3165) was obtained from Sigma.
3
Western Blot and Immunostaining Protocols
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The following antibodies were purchased from following companies and used for Western blotting and immunostaining. Antibodies for USP47 and RPS2 were from Bethyl Laboratories (Montgomery, TX, USA). Antibodies for HA, Myc, p53, and p21 were from Santa Cruz Biotechnology. Antibody for Flag was from Sigma Aldrich (St. Louis, MO, USA). Antibody for MDM2 was from Calbiochem (San Diego, CA, USA). Antibody for β-actin was from Ab Frontier. After Western blotting, the band intensity was measured by Image J and then was normalized by β-actin. The following drugs were purchased from following companies and were treated to cells for the following reasons. MG132 (A.G scientific, San Diego, CA, USA) was treated at a 10 μM concentration to inhibit proteasomal degradation. Actinomycin D (Merck, Kenilworth, NJ, USA) was treated at a 5 nM concentration to induce ribosomal stress. Cycloheximide was treated at a 100 μg/mL concentration to inhibit protein synthesis.
4
Co-Immunoprecipitation of Cell Proteins
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Antibodies for LIN-9 have been described previously [26] (link) and the anti-phosphoT96 was developed by ProteinTech, Inc (Chicago, IL). Antibodies for cyclin E1, cyclin A2 and GFP were from Santa Cruz, antibody for Flag was from Sigma. For IP, cells were lysed in NP-40 lysis buffer (300 mM NaCl, 50 mM Tris pH 8.0, 1 mM MgCl2, 0.2% NP-40, 10% Glycerol, Protease- and Phosphatase-Inhibitors from Roche and Sigma, respectively). For Co-IPs, cell lysis was performed in NP-40 lysis buffer, followed by IP in TIF-buffer (150 mM NaCl, 20 mM Tris pH 8.0, 1 mM MgCl2, 0.1% NP-40, 10% Glycerol and Protease- and Phosphatase-Inhibitors from Roche and Sigma, respectively).
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