Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using PCNA (PC10) Mouse mAb (left) compared to concentration matched Mouse (E5Y6Q) mAb IgG2a Isotype Control (right).
Confocal immunofluorescent analysis of HCT 116 cells using CD44 (156-3C11) Mouse mAb #3570 (left, green) compared to concentration matched Mouse (E5Y6Q) mAb IgG2a Isotype Control (right, green). Red = Propidium Iodide (PI)/RNase Staining Solution #4087.
Flow cytometric analysis of unpermeabilized HeLa cells using CD44 (156-3C11) Mouse mAb #3570 (solid line) compared to concentration-matched Mouse (E5Y6Q) mAb IgG2a Isotype Control (dashed line). Anti-mouse IgG (H+L) F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 mouse embryonic fibroblasts treated with tunicamycin (2ug/ml, 10hr) and either Mouse (E5Y6Q) mAb IgG2a Isotype Control, Normal Rabbit IgG #2729, or CHOP (L63F7) Mouse mAb #2895 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Mouse ATF-3 Intron 1 Primers #13059, mouse CHOP promoter primers, and SimpleChIP® Mouse RPL30 Intron 2 Primers #7015. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Note: This control antibody must be diluted to the same concentration (not dilution) as the specific antibody in analysis. See Directions for Use.
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 mouse IgG2a isotype control are 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 G agarose beads for subsequent 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 October 2016
revised April 2018
Protocol Id: 1244
Mouse (E5Y6Q) mAb IgG2a Isotype Control is not directed against any known antigen. It functions as an isotype control for mouse IgG2a monoclonal 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.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.