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5415
Mouse (G3A1) mAb IgG1 Isotype Control

Mouse (G3A1) mAb IgG1 Isotype Control #5415

APPLICATIONS

Concentration SENSITIVITY Isotype
2.5 mg/ml Endogenous Mouse IgG1, kappa
IHC-P (paraffin)

Immunohistochemical analysis of paraffin-embedded human seminoma using Nanog (1E6C4) Mouse mAb #4893 (left) or Mouse (G3A1) mAb IgG1 Isotype Control (right).

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IF-IC

Confocal immunofluorescent analysis of normal rat cerebellum using Neurofilament H (RMdO 20) Mouse mAb #2836 (green, left) compared to concentration matched Mouse (G3A1) mAb IgG1 Isotype Control (green, right). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IF-IC

Confocal immunofluorescent analysis of HT-29 cells using α-Tubulin (DM1A) Mouse mAb #3873 (green, left) compared to concentration matched Mouse (G3A1) mAb IgG1 Isotype Control (green, right). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Flow Cytometry

Flow cytometric analysis of MCF7 cells using Pan-Keratin (C11) Mouse mAb #4545 (blue) compared to concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control (red).

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Flow Cytometry

Flow cytometric analysis of Jurkat cells, U0126-treated (blue) or TPA-treated (green), using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (E10) Mouse mAb #9106 compared to concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control (red).

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Chromatin IP

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 Mouse (G3A1) mAb IgG1 Isotype Control (red) is compared to the amount of the same DNA sequence enriched by the histone H3-specific immunoprecipitations (blue).

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Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins utilizing Protein G agarose beads for subsequent analysis by western immunoblot or kinase activity.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 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.

  3. 3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer.
  4. Protein G Agarose Beads: (#37478).
  5. 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 μl 10X kinase buffer to 900 μl dH2O, mix.
  6. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 μM), add 10 μl ATP (10 mM) to 490 μl 1X kinase buffer.

B. Preparing Cell Lysates

  1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  2. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS.
  3. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min.
  4. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
  5. Sonicate on ice three times for 5 sec each.
  6. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C.

C. Immunoprecipitation

Cell Lysate Pre-Clearing (Optional)

  1. Vortex to mix beads.
  2. Add 10–30 μl of 50% Protein G agarose bead slurry to 200 μl cell lysate at 1 mg/ml.
  3. Incubate with rotation at 4°C for 30–60 min.
  4. Microcentrifuge for 10 min at 4°C. Transfer the supernatant to a fresh tube.
  5. Proceed to immunoprecipitation below.

Immunoprecipitation

  1. Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 μl cell lysate at 1 mg/ml. Incubate with rotation overnight at 4°C.
  2. Add Protein G agarose (10–30 μl of 50% bead slurry). Incubate with rotation for 1–3 hr at 4°C.
  3. Microcentrifuge for 30 sec at 4°C. Wash pellet five times with 500 μl of 1X cell lysis buffer. Keep on ice between washes.
  4. Proceed to sample analysis by western immunoblotting or kinase activity (section D).

D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting

  1. Resuspend the pellet with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec at 14,000 x g.
  2. Heat the sample to 95–100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g.
  3. Load the sample (15–30 μl) on a 4–20% gel for SDS-PAGE.
  4. Analyze sample by western blot (see Western Immunoblotting Protocol).

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).

For Analysis by Kinase Assay

  1. Wash pellet twice with 500 μl 1X kinase buffer. Keep on ice.
  2. Suspend pellet in 40 μl 1X kinase buffer supplemented with 200 μM ATP and appropriate substrate.
  3. Incubate for 30 min at 30°C.
  4. Terminate reaction with 20 μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec.
  5. Transfer supernatant containing phosphorylated substrate to another tube.
  6. Heat the sample to 95–100°C for 2–5 min and microcentrifuge for 1 min at 14,000 x g.
  7. Load the sample (15–30 μl) on SDS-PAGE (4–20%).

posted October 2016

revised April 2018

Protocol Id: 1244

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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 IgG1 isotype control is used.

Storage:

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.

Mouse (G3A1) mAb IgG1 Isotype Control is not directed against any known antigen. It functions as an isotype control for mouse IgG1 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.

For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.

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