ENCAB000AOD

Antibody against Homo sapiens POLR2AphosphoS5

Homo sapiens
K562, GM12878
characterized to standards
Homo sapiens
vagina, breast epithelium, stomach, HepG2, prostate gland, esophagus muscularis mucosa, upper lobe of left lung, transverse colon, sigmoid colon, induced pluripotent stem cell, bipolar neuron, spleen, HeLa-S3, gastroesophageal sphincter, GM23338, suprapubic skin
characterized to standards with exemption
Status
released
Source (vendor)
Abcam
Product ID
ab5408
Lot ID
648628
Characterized targets
POLR2AphosphoS5 (Homo sapiens)
Lot ID aliases
647708
Host
mouse
Clonality
monoclonal
Antigen description
clone 4H8

Characterizations

POLR2AphosphoS5 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
not reviewed
Caption
IP followed by mass spectrometry: Briefly, K562 whole cell lysates were immunoprecipitated using primary antibody, and the IP fraction was loaded on a 12% acrylamide gel and separated with a Bio-Rad PROTEAN II xi system. Gel was stained with Coomassie Blue in order to visualize marker bands. A gel fragment corresponding to the band indicated above in the western blot image was excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. There the sample was run on an LTQ XL Linear Ion Trap Mass Spectrometer by LC-ESI-MS/MS. Peptides were identified using SEQUEST tandem mass spectra analysis, with probability based matching at p < 0.05. As per ENCODE data standards, all SEQUEST results are attached, including common contaminants. Target protein is listed as hits 3a and 3b. Results in document "ENCODE_HAIB_Pol2-4H8_03182011_MassSpec.pdf"
Submitted by
Richard Myers
Lab
Richard Myers, HAIB
POLR2AphosphoS5 (Homo sapiens)
induced pluripotent stem cellbipolar neuronGM23338
Method: immunoblot
Attachment from submitter
exempt from standards
Caption
The ENCODE Binding Working Group finds for some valuable tissues that recreating a primary on well characterized antibodies is not cost effective. Therefore, they allow exemption from standards for these tissues.
Submitter comment
The lab is asking for an exemption for IPSCs and biopolar spindle neurons (IPSC-derived) cells due to the lack of resources to make a primary characterization for them
Reviewer comment
--
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
POLR2AphosphoS5 (Homo sapiens)
transverse colonstomachsigmoid colonspleensuprapubic skinvaginaprostate glandesophagus muscularis mucosabreast epitheliumgastroesophageal sphincterupper lobe of left lung
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
The ENCODE Binding Working Group finds for some valuable tissues that recreating a primary on well characterized antibodies is not cost effective. Therefore, they allow exemption from standards for these tissues. Compliant characterizations in other cell lines may be used instead.
Submitter comment
The lab is asking for an exemption for transverse colon and stomach tissue due to the lack of resources to make a primary characterization for them
Reviewer comment
--
Submitted by
Aditi Narayanan
Lab
Richard Myers, HAIB
Download
POLR2AphosphoS5 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
compliant
Caption
K562 whole cell lysate was immunoprecipitated using the primary antibody (Abcam; 5408). The IP fraction was loaded on a 12% Bio-Rad TGX gel and separated with the Bio-Rad Tetra Cell system. A gel fragment (rectangle outline) corresponding to the band indicated on the Coomassie Blue stained gel image was excised and sent to the University of Alabama at Birmingham Cancer Center Mass Spectrometry/Proteomics Shared Facility. Analysis of gel fragment 1 from K562: The sample was analyzed on a LTQ XL Linear Ion Trap Mass Spectrometer by LC-ESI-MS/MS. Peptides were identified using SEQUEST tandem mass spectral analysis with probability based matching at p < 0.05. SEQUEST results were reported with ProteinProphet protXML Viewer (TPP v4.4 JETSTREAM) and filtered for a minimum probability of 0.9. All protein hits that met these criteria were reported, including common contaminants. Fold enrichment for each protein reported was determined using a custom script based on the FC-B score calculation from the reference Mellacheruvu et al., 2013. The CRAPome: a contaminant repository for affinity purification mass spectrometry data. Nat. Methods. 10(8):730-736. Doi:10.1038/nmeth.2557. The target protein, POLR2A/RPB1, was identified as the 2nd enriched protein and the 1st polymerase based on IP-Mass Spectrometry.
Submitter comment
In some cases, we exercised the option of extracting the whole lane to send for IP-mass spectrometry as opposed to cutting and sending discrete bands from a gel. This is especially the case when the antibody results in multiple and non-discrete bands or no visible bands in the predicted size range of the protein when observed by IP-Western. To ensure that the targeted protein can be identified in the IP, whole cell lysates are loaded in a 12% Bio-Rad TGX gel and run for 5-10 minutes using the Bio-Rad Tetra Cell system. The entire gel lane or just the single unseparated band is excised from the gel and sent for analysis. We do not provide coomassie blue-stained gel images in these instances as all one observes is a thick, unresolved band near the loading well.
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB
Download
POLR2AphosphoS5 (Homo sapiens)
K562GM12878HepG2HeLa-S3
Method: immunoblot
Attachment from submitter
compliant
Caption
Whole cell lysates of K562, GM12878, HepG2, and HeLa were immunoprecipitated using the primary antibody (Abcam; 5408). The IP fraction was separated on a 12% acrylamide gel with the Bio-Rad PROTEAN II xi system. After separation, the samples were transferred to a nitrocellulose membrane with an Invitrogen iBlot system. The membrane was probed with the primary antibody (same as that used for IP) and a secondary HRP-conjugated antibody. The resulting bands were visualized with SuperSignal West Femto Solution (Thermo Scientific). Protein Marker (PM) is labeled in kDa. A single prominent band was detected at ~230 kDa. Whole cell lysates of K562, GM12878, HepG2, and HeLa were immunoprecipitated using the primary antibody (Abcam; 5408). The IP fraction was separated on a 12% acrylamide gel with the Bio-Rad PROTEAN II xi system. After separation, the samples were transferred to a nitrocellulose membrane with an Invitrogen iBlot system. The membrane was probed with the primary antibody (same as that used for IP) and a secondary HRP-conjugated antibody. The resulting bands were visualized with SuperSignal West Femto Solution (Thermo Scientific). Protein Marker (PM) is labeled in kDa. A single prominent band was detected at ~230 kDa. Expected size ~217 kDa
Submitted by
Mark Mackiewicz
Lab
Richard Myers, HAIB