Meerschweinchen anti-Kaninchen IgG (Heavy & Light Chain) Antikörper - Preadsorbed

Produktnummer ABIN101961

Produktdetails

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

Target: IgG

Bindungsspezifität: Heavy & Light Chain

Reaktivität: Kaninchen

Wirt: Meerschweinchen

Klonalität: Polyklonal

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

Verwendungszweck:

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).

Spezifität: Rabbit IgG (H&L)

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

Aufreinigung: Preadsorption: Solid phase absorption

Sterilität: Sterile filtered

Immunogen: whole molecule of rabbit IgG

Isotyp: IgG

Anwendungsinformationen

Applikationshinweise: 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

Kommentare:

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.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Independent Validation (CUT&Tag)

Validierung #104174 (Cleavage Under Targets and Tagmentation)

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

No.: #104174

Datum: 18.12.2019

Antigen: rabbit IgG

Chargennummer: 43586

Validierte Anwendung: Cleavage Under Targets and Tagmentation

Positivkontrolle:

rabbit anti-H3K27me3 monoclonal antibody

Negativkontrolle:

rabbit normal IgG antibody

Bewertung:

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.

Validierungsbilder

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.

Protokoll

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

Sekundärantikörper: 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).

Anmerkungen:

• 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.

Handhabung

Format: Liquid

Konzentration: 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

Konservierungsmittel: Sodium azide

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

Lagerung: 4 °C,-20 °C

Haltbarkeit: 12 months

Referenzen

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Antigendetails

Target: IgG

Abstract: IgG Produkte

Substanzklasse: Antibody