1. Antibodies
  2. Homo sapiens + Mus musculus
  3. POLR2A + Polr2a

ENCAB000AOB

Antibody against Homo sapiens POLR2AphosphoS2, Mus musculus POLR2AphosphoS2

Homo sapiens
K562
characterized to standards
Homo sapiens
any cell type or tissue
partially characterized
Mus musculus
any cell type or tissue
partially characterized
Status
released
Source (vendor)
Abcam
Product ID
ab5095
Lot ID
GR32890-1
Host
rabbit
Clonality
polyclonal
Purification
affinity
Antigen description
Raised against peptide conjugated to KLH derived from within residues 1600 - 1700 of S. cerevisiae RNA polymerase II CTD repeat YSPTSPS, phosphorylated at S2.

Characterizations

POLR2AphosphoS2 (Homo sapiens)
Method: immunoprecipitation
Attachment PDF Icon
not reviewed
Caption
Immunoprecipitation was performed on nuclear lysates from K562 cells using antibody ab5095 against PolIIS2. Lane1: Nuclear lysate. Lane 2: Unbound material from immunoprecipitation with ab5095. Lane 3: Bound material from immunoprecipitation with ab5095. Lane 4: Bound material from control immunoprecipitation with rabbit IgG. Arrow indicates band of expected size (240 kD) that is enriched in the specifically immunoprecipitated fraction. Smaller band detected in the IP is possibly degradation product as indicated by the Mass Spec analysis. Immunoprecipitation of K562 nuclear lysate enriches a protein of ~240 KD.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
POLR2AphosphoS2 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
not reviewed
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
POLR2AphosphoS2 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
compliant
Caption
Immunoprecipitation of PolIIS2 from K562 cell using ab5095 antibody. Lane 1: input nuclear lysate. Lane 2: material immunoprecipitated with ab5095. Lane 3: material immunoprecipitated using control IgG. Bands A and B were excised from the gel and subjected to analysis by mass spectrometry.
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
POLR2AphosphoS2 (Mus musculus)
Method: immunoprecipitation
Attachment PDF Icon
not reviewed
Caption
Immunoprecipitation of PolIIS2 from K562 cells using ab5095 antibody. Lane 1: input nuclear lysate, Lane 2: material immunoprecipitated with ab5095, Lane 3: material immunoprecipitated using control IgG. Bands A and B were excised from the gel and subjected to analysis by mass spectrometry. IP followed by masspectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using ab5095, 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 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). We report 38 proteins identified in band A, although 11 of these are also present in a control immunoprecipitation and are thus likely to present due to non-specific association with the IP matrix. Of the specifically immunoprecipitated proteins, GRp20 is the most abundant (99 peptides). Based on these observations, this band is likely due to the presence of immunoprecipitated GRp20 and Sc-1002 meets the ENCODE standard for validation by this criterion.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
POLR2AphosphoS2 (Mus musculus)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
not reviewed
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
POLR2AphosphoS2 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
compliant
Caption
IP followed by mass spectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using ab5095 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 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 discover rate <1%, 2 peptides per protein minimum).
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford