Cobaye anti-Lapin IgG (Heavy & Light Chain) Anticorps - Preadsorbed

N° du produit ABIN101961

Détail du produit

Highlights:

• 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

Antigène: IgG

Épitope: Heavy & Light Chain

Reactivité: Lapin

Hôte: Cobaye

Clonalité: Polyclonal

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

Fonction:

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).

Specificité: Rabbit IgG (H&L)

Réactivité croisée (Details): No reaction was observed against Human, Mouse and Goat Serum Proteins.

Purification: Preadsorption: Solid phase absorption

Stérilité: Sterile filtered

Immunogène: whole molecule of rabbit IgG

Isotype: IgG

Information d'application

Indications d'application: 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

Commentaires:

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: 18.12.2019

Antigène: rabbit IgG

Numéro du lot: 43586

Application validée: Cleavage Under Targets and Tagmentation

Contrôle positif:

rabbit anti-H3K27me3 monoclonal antibody

Contrôle négative:

rabbit normal IgG antibody

Conclusion:

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.

Protocole

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

Anticorps secondaire: 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).

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.

Stockage

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

Agent conservateur: Sodium azide

Précaution d'utilisation: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Stock: 4 °C,-20 °C

Date de péremption: 12 months

Référence

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Détail du antigène

Antigène: IgG

Abstract: IgG Produits

Classe de substances: Antibody