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 Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag)

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.

Independent Validation (CUT&RUN)

Validation #104622 (Cleavage Under Targets and Release Using Nuclease)

by: Cantù Lab, Gene Regulation during Development and Disease, Linköping University

No.: #104622

Date: 02/10/2024

Antigen: IgG

Lot Number: 48661

Method validated: Cleavage Under Targets and Release Using Nuclease

Positive Control:

Anti H3K4me (antibodies-online, ABIN3023251)

Negative Control:

Guinea Pig anti-rabbit IgG (antibodies-online, ABIN101961)

Notes:

Passed.

Validation Images

Library profiles comparing fragment size distributions on an E-Gel EX 2% agarose gel (Thermo Fisher). Fragments obtained from CUT&RUN using ABIN6923144 (left) after library preparation, compared to the E-Gel Sizing DNA Ladder (Thermo Fisher) (right).

Alignment tracks from CUT&RUN in human fibroblast cells using ABIN101961 IgG antibody showing the WT1. 2. RefSeq Genes.

Full Methods

Primary Antibody: ABIN101961

Full Protocol:

• Cell harvest and nuclear extraction
  • Harvest 250,000 HEK293T cells per antibody
  • Centrifuge cell solution 5 min at 600 x g at RT.
  • Remove the liquid carefully.
  • Gently resuspend cells in 2 mL of Nuclear Extraction Buffer (20 mM HEPES-KOH pH 8.2, 20% Glycerol, 0.05% IGEPAL, 0.5 mM Spermidine, 10 mM KCl, Roche Complete Protease Inhibitor EDTA-free).
  • Move the solution to a 2 mL centrifuge tube.
  • Pellet the nuclei 800 x g for 5 min.
  • Repeat the NE wash twice for a total of three washes.
  • Resuspend the nuclei in 40 µL NE Buffer per sample.
• Concanavalin A beads preparation
  • Prepare one 2 mL microcentrifuge tube.
  • Gently resuspend the magnetic Concanavalin A Beads (antibodies-online, ABIN6923139).
  • Pipette 10 µL Con A Beads slurry for each sample into the 2 mL microcentrifuge tube.
  • Place the tube on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Remove the microcentrifuge tube from the magnetic stand.
  • Pipette 1 mL Binding Buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) into the tube and resuspend ConA beads by gentle pipetting.
  • Spin down the liquid from the lid with a quick pulse in a table-top centrifuge.
  • Place the tubes on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Repeat the wash twice for a total of three washes.
  • Gently resuspend the ConA Beads in 44 µL Binding Buffer.
• Nuclei immobilization – binding to Concanavalin A beads
  • Carefully vortex the nuclei suspension and add 44 µL of the Con A beads in Binding Buffer to the cell suspension for each sample.
  • Close tube tightly and incubate 10 min at 4 °C.
  • Put the 2 mL tube on the magnet stand and when the liquid is clear, remove the supernatant.
  • Resuspend the beads in 1 mL of EDTA wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free, 2 mM EDTA).
  • Incubate 5 min at RT.
  • Resuspend the beads in 200 µL of wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, Roche Complete Protease Inhibitor EDTA-free).
• Primary antibody binding
  • Divide nuclei suspension into separate 200 µL PCR tubes, one for each antibody.
  • Add 2 µL antibody (anti-H3K4me positive control ABIN3023251, and guinea pig anti-rabbit IgG negative control antibody ABIN101961) to the respective tube, corresponding to a 1:100 dilution.
  • Incubate at 4 °C overnight.
  • Wash with 200 µL of Wash Buffer using a multichannel pipette to accelerate the process.
  • Repeat the wash five times for a total of six washes.
• pAG-MNase Binding
  • Prepare a 1.5 mL microcentrifuge tube containing 200 µL of pAG mix per sample (200 µL of wash buffer + 120 ng pAG-MNase per sample).
  • Place the PCR tubes with the sample on a magnet stand until the fluid is clear. Remove the liquid carefully.
  • Resuspend the beads in 200 µL of pAG-MNase premix.
  • Incubate 30 min at 4 °C.
  • Wash with 200 µL of Wash Buffer using a multichannel pipette to accelerate the process.
  • Repeat the wash five times for a total of six washes.
  • Resuspend in 100 µL of Wash Buffer.
• MNase digestion and release of pAG-MNase-antibody-chromatin complexes
  • Place PCR tubes on ice and allow to chill.
  • Prepare a 1.5 mL microcentrifuge tube with 102 µL of 2 mM CaCl2 mix per sample (100 µL Wash Buffer + 2 µL 100 mM CaCl2) and let it chill on ice.
  • Resuspend the sample in 50 µL of 1x Urea STOP Buffer (8.5 M Urea, 100 mM NaCl, 2 mM EGTA, 2 mM EDTA, 0.5% IGEPAL).
  • Incubate the samples 1h at 4 °C.
  • Transfer the supernatant containing the pAG-MNase-bound digested chromatin fragments to fresh 200 µL PCR tubes.
• DNA Clean up (Mag-Bind® TotalPure NGS - M1378-01)
  • Add 2x volume of beads to each sample (e.g. 100 µL of beads for 50 µL of sample).
  • Wash with 80% EtOH.
  • Resuspend the beads in 20 µL of 10 mM Tris.
• Library preparation and sequencing
  • Prepare Libraries using KAPA HyperPrep Kit using KAPA Dual-Indexed adapters according to protocol.
  • Sequence samples on an Illumina NextSeq 500 sequencer, using a NextSeq 500/550 High Output Kit v2.5 (75 Cycles), 36bp PE.
• Peak calling
  • Trim reads using bbTools bbduk to remove adapters, artifacts, and repeat sequences.
  • Aligned reads were mapped to the hg38 human genome using bowtie with options -m 1 -v 0 -I 0 -X 500.
  • Use SAMtools to convert SAM files to BAM files and remove duplicates.
  • Use BEDtools genomecov to produce Bedgraph files.
  • Call peaks using SEACR with a 0.001 threshold and the option norm stringent.

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