Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858 (left) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control (right).
Confocal immunofluorescent analysis of A549 cells using β-Tubulin (9F3) Rabbit mAb #2128 (green, left) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control (green, right). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Flow cytometric analysis of MCF7 cells (blue) and HeLa cells (green) using Vimentin (D21H3) XP® Rabbit mAb #5741 (solid lines) or a concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Chromatin immunoprecipitations were performed using digested chromatin from HeLa cells and the indicated antibodies. Purified DNA was analyzed by quantitative real-time PCR, using SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human RPL30 Exon 3 Primers #7014, and SimpleChIP® Human MYT-1 Exon 1 Primers #4493. The relative abundance of each DNA sequence enriched by Rabbit (DA1E) mAb IgG XP® Isotype Control (red) is compared to the amount of the same DNA sequence enriched by the histone H3-specific immunoprecipitations (blue).
Important! Dilute this control antibody to the same concentration (not dilution) as the specific antibody used for analysis. Higher background may result if excessive amounts of rabbit IgG isotype control is used.
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
This protocol is intended for immunoprecipitation of native proteins utilizing Protein A agarose beads for analysis by western immunoblot or kinase activity.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.
NOTE: Add 1 mM PMSF (#8553) immediately prior to use.
Proceed to one of the following specific set of steps.
NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (L57A3) mAb (#3677) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).
posted December 2008
revised April 2018
Protocol Id: 409
Rabbit (DA1E) mAb IgG XP® Isotype Control is not directed against any known antigen. It functions as an isotype control for rabbit IgG antibodies.
Isotype control antibodies are used to estimate the nonspecific binding of target primary antibodies due to Fc receptor binding or other protein-protein interactions. An isotype control antibody should have the same immunoglobulin type and be used at the same concentration as the test antibody.
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