ENCAB000AOC

Alternate accession: ENCAB000BSG

Antibody against Homo sapiens POLR2A, Mus musculus POLR2A

Homo sapiens
GM12892, GM12878, lower leg skin, body of pancreas, K562, HeLa-S3, GM12891
characterized to standards with exemption
Homo sapiens
any cell type or tissue
partially characterized
Mus musculus
any cell type or tissue
Status
released
Source (vendor)
Covance
Product ID
MMS-126R
Lot ID
14861301
Lot ID aliases
14922701, E11BF00348
Host
mouse
Clonality
monoclonal
Purification
crude
Antigen description
clone 8WG16

Characterizations

POLR2A (Homo sapiens)
GM12891GM12892body of pancreaslower leg skin
Method: immunoblot
Attachment from submitter
exempt from standards
Caption
Primary characterization (e.g. immunoblot, immunoprecipitation) for the specified cell types and tissues are exempted for this antibody by the ENCODE antibody review panel because it is a well-characterized monoclonal antibody that has been in use without issue by the community for many years.
Submitter comment
This is a well characterized antibody already used in many ENCODE experiments.
Reviewer comment
Primary characterizations for this antibody were exempted for all cell types and tissues by the Antibody Review Panel on March 30, 2016.
Submitted by
Esther Chan
Lab
J. Michael Cherry, Stanford
Download
POLR2A (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
not reviewed
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
POLR2A (Homo sapiens)
Method: immunoprecipitation
not reviewed
Caption
A single band consistent with the mobility expected for POLR2A/RPB1 (~217kD) is observed in nuclear extracts from cell lines K562 and HeLa S3 (Panel A) and cell line GM12878 (Panel B). This band is efficiently and specifically immunoprecipitated from GM12878 nuclear extract by antibody MMS-126R (Panel B). Analysis by mass spectrometry of material immunoprecipitated by MMS-126R from HeLaS3 cells shows very strong enrichment for POLR2A and associated components of RNA polymerase II (see validation assay #2). Therefore, MMS-126R meets this criterion for validation.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
POLR2A (Homo sapiens)
GM12878
Method: immunoprecipitation
Attachment from submitter
compliant
Caption
Immunoprecipitation: GM12878 nuclear extract was immunoprecipitated with 1,2 and 4ug Pol II antibody (Covance, MMS‐126R) or normal mouse IgG as a negative control. Supernatant was checked to see the completion of IP. Arrow indicates band specifically immunoprecipitated by MMS‐126R (expected band size ~217kD). The lane found compliant is lane 9, labelled "Pol II IP, 4ug".
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
POLR2A (Mus musculus)
Method: immunoprecipitation followed by mass spectrometry
not reviewed
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
POLR2A (Mus musculus)
Method: immunoprecipitation
not reviewed
Caption
Immunoprecipitation of POLR2A from HeLa S3 cells using MMS-126R followed by mass spectrometry: Briefly, HeLaS3 whole cell lysates were immunoprecipitated using MMS-126R ,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 the full length of the gel were excised and destained using the SilverSNAP Stain for Mass Spectrometry (Pierce). Then proteins were trysinized using the in-gel digestion method. Digested proteins were analyzed on an LTQ-Orbitrap (Thermo Scientific) by 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). Samples were run twice and results from both runs are presented We report 158 proteins identified in this immunoprecipitation. However, only 21 of these were identified in the immunoprecipitation with MMS-126R, but not the control immunoprecipitation. Of these 21, POLR2A is the most abundant (highlighted in yellow) and all other abundantly identifed proteins (highlighted in blue) are known to be part of the RNA polymerase II complex . Based on these observations, MMS-126R meets the ENCODE standard for validation by this criterion.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
POLR2A (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
exempt from standards
Caption
Immunoprecipitation of POLR2A from HeLa-S3 cells using MMS-126R followed by mass spectrometry. Briefly, HeLa-S3 whole cell lysates were immunoprecipitated using MMS-126R, 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 full length of the gel were excised and destained using the SilverSNAP Stain for Mass Spectrometry (Pierce). Then, proteins were trypsinized using the in-gel digestion method. Digested proteins were analyzed on an LTQ-Orbitrap (Thermo Scientific) by 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). Samples were run twice and results from both runs are presented.
Submitter comment
This characterization was done a long time ago and an image of the IP before mass-spec and the full mass-spec peptide table are no longer available to submit.
Reviewer comment
The lack of an image of the IP with the excised band(s) marked was OK'd by the ENCODE antibody review panel (03/28/16).
Submitted by
Minyi Shi
Lab
Michael Snyder, Stanford
POLR2A (Homo sapiens)
K562HeLa-S3
Method: immunoblot
Attachment from submitter
compliant
Caption
Immunoblotting: A single band consistent with the mobility expected for POLR2A/RPB1 (~217kD) is observed in nuclear extracts from cell lines K562 and HeLa S3 and cell line GM12878. This band, labeled by the arrow, is efficiently and specifically immunoprecipitated from GM12878 nuclear extract by antibody MMS-126R.
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford