ENCAB529YRC

Antibody against Homo sapiens RAD21

Homo sapiens
neural progenitor cell, SU-DHL-2, GM12878, K1, KU-19-19, hepatocyte, MSiPS, hTERT-HME1, pulmonary artery endothelial cell, HT-1197, MSLCL, DU 145, Jurkat clone E61, ARPE-19, H9, LNCAP, fibroblast, HT-1376, LNCaP clone FGC, NCI-H1437, neural cell, HepG2, SU-DHL-4, K562, MCF-7
characterized to standards with exemption
Status
released
Source (vendor)
Abcam
Product ID
ab992
Lot ID
GR184716
Characterized targets
RAD21 (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Purification
Protein G
Isotype
IgG
Antigen description
Synthetic peptide (Human) conjugated to KLH - which represented a portion of human Rad21 encoded within exon 14
Aliases
michael-snyder:AS-1645
External resources

Characterizations

RAD21 (Homo sapiens)
Method: knockdown or knockout
Attachment from submitter
exempt from standards
Caption
A new secondary characterization is not required as one exists for ENCAB000AKG which is a lot of the same antibody (ab992). The primary characterization (https://www.encodeproject.org/antibody-characterizations/9075a02c-f1f5-4456-965f-a10322163710/) of K562 for this lot shows the same banding pattern as the primary characterization (https://www.encodeproject.org/antibody-characterizations/a998bebe-57f7-4de9-8732-735fd07a8764) in K562 cell line for the lot ENCAB000AKG.
Submitter comment
This is a new lot of a previously characterized antibody ENCAB000AKG and we think no further characterization is necessary beyond the primary if the banding pattern is consistent with the old.
Reviewer comment
The banding pattern in K562, GM12878 and HepG2 is consistent with that of the primary characterizations associated with old lot ENCAB000AKG so a new secondary characterization for the new lot is not needed
Submitted by
Jessika Adrian
Lab
Michael Snyder, Stanford
RAD21 (Homo sapiens)
neural cellfibroblastHepG2K562MCF-7GM12878LNCaP clone FGCSU-DHL-2Jurkat clone E61K1ARPE-19DU 145NCI-H1437HT-1376hTERT-HME1LNCAPpulmonary artery endothelial cellH9KU-19-19HT-1197SU-DHL-4neural progenitor cellhepatocyteMSiPSMSLCL
Method: immunoblot
Attachment from submitter
compliant
Caption
Western blot analysis of nuclear lysates prepared from multiple cells lines loaded in the order: HepG2, K562, MCF-7, GM12878, LNCaP, SU-DHL-2, Jurkat using the antibody ab992. Molecular Weight: 75
Submitter comment
While the predicted size of Rad21 is ~72kD, according to product characterization literature (found at manufacturer website), the bands at ~70kD are of unknown origin while a band of ~140kD, similar to the one observed here, is identified as Rad21. In support of this, siRNAs targeting Rad21 dramatically reduce levels of the 140kD band, while levels of the 70kD band are unaffected (see Figure 2 of this document). 1. Hyperphosphorylation (ref. PMID 11073952 & many others) https://www.ncbi.nlm.nih.gov/gene/5885 2. Dimerisation as part of cohesin complex (whose other members we can detect in the 130kDa band, refs. PMIDs 23874961 & 19075111)
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford
RAD21 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Caption
IP followed by mass spectrometry. Briefly, protein was immunoprecipitated from K562 nuclear cell lysates using the antibody ab992, and the IP fraction was loaded on a 10% polyacrylamide gel (NuPAGEBis-Tris Gel) and separated with an Invitrogen NuPAGE electrophoresis system. The gel was stained by ColloidialCoomassie G-250 stain, gel fragments corresponding to the bands indicated were excised. Then proteins were trypsinized using the in-gel digestion method. Digested proteins were analyzed on an Orbitrap Elite mass spectrometer (Thermo Scientific) by the nanoLC-ESI-MS/MS technique. Peptides were identified by the SEQUEST algorithm and filtered with a high confidence threshold (Peptide false discovery rate < 1%, 2 unique peptides per protein minimum, mass error < 10 ppm).
Submitter comment
Band B: RAD21 peptides could not be detected, however siRNA treatment results in knockdown of Band B (see antibody lot ENCAB000AKG). Hyperphosphorylation of RAD21 or dimerisation as part of cohesin complex might result in a more stable version of the protein which couldn’t be fragmented during our mass spec analysis.
Reviewer comment
There is clearly some pattern of RAD21 affecting the upper bands even though the Mass Spec does not find isolated RAD21. The siRNA knockdown found in ENCAB000AKG is evidence of this.
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford
RAD21 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
compliant
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line K562 using the antibody ab992. Lane 1: input nuclear lysate. Lane 2: material immunoprecipitated with antibody. Lane 3: material immunoprecipitated using control IgG. Marked bands were excised from gel and subjected to analysis by mass spectrometry. Target molecular weight: 75.
Submitter comment
While the predicted size of Rad21 is ~72kD, according to product characterization literature (found at manufacturer'€™s website), the bands at ~70kD are of unknown origin while a band of ~140kD, similar to the one observed here, is identified as Rad21. In support of this, siRNAs targeting Rad21 dramatically reduce levels of the 140kD band, while levels of the 70kD band are unaffected (see Figure 2 of this document). 1. Hyperphosphorylation (ref. PMID 11073952 & many others) https://www.ncbi.nlm.nih.gov/gene/5885 2. Dimerisation as part of cohesin complex (whose other members we can detect in the 130kDa band, refs. PMIDs 23874961 & 19075111)
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford