ENCAB000ASP

Antibody against Homo sapiens ARID3A

Homo sapiens
GM12878, K562, MCF-7
characterized to standards
Homo sapiens
any cell type or tissue
partially characterized
Status
released
Source (vendor)
Novus
Product ID
NB100-279
Lot ID
A1
Characterized targets
ARID3A (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Purification
affinity
Antigen description
Immunogen: A synthetic peptide, which represented a portion of human Dead Ringer-Like 1 encoded within exon 8.

Characterizations

ARID3A (Homo sapiens)
GM12878
Method: immunoprecipitation
Attachment from submitter
compliant
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line: GM12878 using the antibody NB100-279. The image shows western blot analysis of input, flowthrough, immunoprecipitate, and mock immunoprecipitate using IgG. Target molecular weight: 80.0.
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford
ARID3A (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
compliant
Caption
IP followed by mass spectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using NB100-279, and the IP fraction was loaded on a 10% polyacrylamide gel (NuPAGE Bis-Tris Gel) and separated with an Invitrogen NuPAGE electrophoresis system. The gel was silver-stained, gel fragments corresponding to the bands indicated were excised and destained using the SilverSNAP Stain for Mass Spectrometry (Pierce). Then proteins were treated with trypsin using the in-gel digestion method. Digested proteins were analyzed on a LTQ-Orbitrap (Thermo Scientific) b the nanoLC-ESI-MS/MS technique. Peptides were identified by the SEQUEST algorithm and filtered with a high confidence threshold (protein false discovery rate <1%, 2 peptides per protein minimum).
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
ARID3A (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
compliant
Caption
Immunoprecipitation of ARID3A from K562 cells using NB100-279. Lane 1: input nuclear lysate. Lane 2: material immunoprecipitated with NB100-279. Lane 3: material immunoprecipitated using control IgG. Band A was excised from gel and subject to analysis by mass spectrometry. Expected band size is ~80kD.
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
Download
ARID3A (Homo sapiens)
MCF-7
Method: immunoprecipitation
Attachment from submitter
compliant
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line: MCF-7, using the antibody NB100-279. The blot shows western blot analysis of input, flowthrough, immunoprecipitate and mock immunoprecipitate using IgG.
Submitted by
Denis Salins
Lab
Michael Snyder, Stanford
ARID3A (Homo sapiens)
Method: immunoprecipitation
not reviewed
Caption
Using antibody NB100-279, we observe a main band in Western blots on lysates from cell lines K562 and HepG2 at a position consistent with a molecular weight of ~75kD (Panel A). This band is efficiently and specifically immunoprecipitated from K562 nuclear extracts (Panel B). This mobility is somewhat slower than expected given the predicted size of ARID3A (~63kD) but is consistent with the electrophoretic mobility reported for ARID3A (see product literature for both antibodies and note the consistent mobility observed with each antibody. A band of ~75kD is also observed in Western blots on lysates from cell lines K562 using antibody sc8821 (Panel C). Western blotting with this antibody was made difficult by high background signal consistently observed on these blots (a problem routinely encountered with antibodies raised in goats from this manufacturer). To verify that this band was ARID3A, we used sc8821 to perform an immunoprecipitation and then used NB100-279 in a Western blot to analyze immunoprecipitated material (Panel D). We observed a single band immunoprecipitated from K562 cells at high efficiency that is consistent with the observed mobility of ARID3A. We conclude this antibody specifically recognizes ARID3A. Based on these data, both antibodies validated by this criterion.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
ARID3A (Homo sapiens)
Method: ChIP-seq comparison
not reviewed
Caption
Because the overlap of the top 40% of peaks from datasets derived from antibodies NB100-279 and sc-8821 (compared using standard ENCODE scoring methods) exceeds the recommended threshold of 80%, both antibodies meet this criterion for validation.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford