Guinea Pig anti-Rabbit IgG (Heavy & Light Chain) Antibody - Preadsorbed

Catalog No. ABIN101961

Product Details

Key Features:

• 95+ publication references, 1 independent validation
• Frequently used as CUT&RUN IgG negative control and CUT&Tag secondary antibody
• Used in CUT&RUN and CUT&Tag protocols, e.g. Henikoff et al. (2018) PMID 29651053, Kaya-Okur et al. (2019, 2020) PMID 31036827 and PMID 32913232

Target: IgG

Binding Specificity: Heavy & Light Chain

Reactivity: Rabbit

Host: Guinea Pig

Clonality: Polyclonal

Application: ELISA, Immunohistochemistry (IHC), Western Blotting (WB), Cleavage Under Targets and Tagmentation (CUT&Tag), Cleavage Under Targets and Release Using Nuclease (CUT&RUN)

Purpose:

The Guinea Pig anti-Rabbit IgG antibody ABIN101961 is well suited as a CUT&RUN IgG negative control and as a secondary antibody in CUT&Tag. It is a component of all our CUT&RUN Product Sets.

Find more products for CUT&RUN and CUT&Tag:

• Overview of CUT&RUN Antibodies.
• Magnetic Concanavalin A beads (agarose) for CUT&RUN (ABIN6952467).
• CUTANA™ pAG-MNase for ChIC/CUT&RUN Assays (ABIN6950951).

Specificity: Rabbit IgG (H&L)

Cross-Reactivity (Details): No reaction was observed against Human, Mouse and Goat Serum Proteins.

Purification: Preadsorption: Solid phase absorption

Sterility: Sterile filtered

Immunogen: whole molecule of rabbit IgG

Isotype: IgG

Application Details

Application Notes: The guinea pig anti-rabbit IgG antibody ABIN101961 is suitable for use in ELISA, immunohistochemistry, and Western Blot, CUT&RUN and CUT&Tag. Specific conditions for each assay should be optimized by the end user. General ABIN101961 dilution recommendations for different applications are as follows:

• ELISA: 1:20,000 - 1:100,000
• WB: 1:2,000 - 1:10,000
• IHC: 1:1,000 - 1:5,000
• CUT&RUN: 1:100
• CUT&Tag: 1:100

Comment:

ABIN101961 is tested via ELISA to ensure that the titer against the antigen (Rb IgG) is above a certain threshold. We also test to make sure the titer against potentially cross-reactive human IgG, goat IgG, and mouse IgG is below a certain threshold.
In addition, we test ABIN101961 against anti-guinea pig Serum, rabbit IgG, and rabbit serum in an immunoelectrophoresis assay.

Restrictions: For Research Use only

Independent Validation (CUT&Tag)

Validation #104174 (Cleavage Under Targets and Tagmentation)

by: Tom Taghon’s lab, Vakgroep Diagnostische Wetenschappen, Universiteit Gent

No.: #104174

Date: 12/18/2019

Antigen: rabbit IgG

Lot Number: 43586

Method validated: Cleavage Under Targets and Tagmentation

Positive Control:

rabbit anti-H3K27me3 monoclonal antibody

Negative Control:

rabbit normal IgG antibody

Notes:

Passed. ABIN101961 successfully increased the number of protein A binding sites in a CUT&Tag protocol on human primary thymocytes and PER-117 cells using a monoclonal rabbit H3K27me3 primary antibody.

Validation Images

CUT&Tag library generated from thymocytes using a monoclonal rabbit anti-H3K27me3 primary antibody and ABIN101961 as a secondary antibody.

CUT&Tag library generated from PER-117 cells using a monoclonal rabbit anti-H3K27me3 primary antibody and ABIN101961 as a secondary antibody.

Full Methods

Primary Antibody: rabbit IgG anti-H3K27me3 antibody (Cell Signaling Technology, 9733T, lot 14)

Secondary Antibody: ABIN101961

Full Protocol:

• Profiling of H3K27me3 signals in human primary CD34+ sorted thymocytes and PER-117 cell line (50,000 cells each).
• Carry out the CUT&Tag protocol according to the single-tube bench top protocol for CUT&Tag developed by Steven Henikoff’s lab as outlined on the protocols.io platform.
• Use reagents as suggested in the original protocol. The pA-Tn5 fusion protein used in this validation was a courtesy of Steven Henikoff’s lab and used at 1:1000 dilution for 100 µL per sample.
• Primary antibody binding with
• • 1µl/sample monoclonal rabbit IgG anti-H3K27me3 antibody (Cell Signaling Technology, 9733T, lot 14) or
  • 1µl/sample rabbit normal IgG (Cell Signaling, 2729S, lot 9).
• Secondary antibody binding with 1µl/sample guinea pig anti rabbit antibody (antibodies-online, ABIN101961, lot 43586).
• Determine library concentration on a Qubit Fluorometer using Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Scientific, Q32581).
• Determine library size distribution of all samples on an Agilent Fragment Analyzer using High Sensitivity Small DNA Fragment Analysis Kit (Agilent, DNF-477-0500).

Experimental Notes:

• ABIN101961 successfully increased the number of protein A binding sites for each bound rabbit anti-H3K27me3 antibody in the human primary thymocytes and PER-117 cell line. This resulted in quantifiable amounts of tagmented genomic fragments after PCR amplification that showed a ladder-like distribution.

• Library concentrations as measured on a Qubit Fluorometer were 0.122ng/µl and 0.538ng/µl for thymocytes and PER-117 cells respectively using a rabbit H3K27me3 primary antibody. Library concentration for both sample was below the instrument’s detections limit using the rabbit normal IgG primary antibody.

• As discussed at the protocols.io, it is expected to observe a ladder-like distribution for H3K27me3 profiling. However, using lower cell numbers could alter the nucleosomal patterns and result in an increase in larger fragments. This is observed by Steven Henikoff’s lab as well. Nevertheless, it does not affect the quality of the sequencing results.

Handling

Format: Liquid

Concentration: 1.02 mg/mL

Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, 0.01 % (w/v) NaN3, no stabilizer

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: 4 °C,-20 °C

Expiry Date: 12 months

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Target Details

Target: IgG

Abstract: IgG Products

Target Type: Antibody