1. Antibodies
  2. Homo sapiens
  3. EP400

ENCAB702SYV

Antibody against Homo sapiens EP400

Homo sapiens
K562
characterized to standards with exemption
Status
released
Source (vendor)
Bethyl Labs
Product ID
A300-541A
Lot ID
1
Characterized targets
EP400 (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Purification
affinity
Aliases
michael-snyder:AS-1482
External resources

Characterizations

EP400 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
exempt from standards
Caption
IP followed by mass spectrometry. Briefly, protein was immunoprecipitated from K562 nuclear cell lysates using the antibody A300-541A, 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
None of the proteins with 2 or more than 2 peptide counts in band A, B and C have been shown to be sequence-specific TFs (including TRRAP, TR150, HNRL1, EPC2, ZC3H11A, ARID1A, SMARCC2, TNRC6B, ZC3H11A, FUS, TOX4, TAF15). No proteins detected in band D binding DNA with sequence-specificity (including FUS, TAF15, HNRNPD, HNRNPH1).
Reviewer comment
The target factor is a co-activator that interacts with, possibly, hundreds of other proteins so I wouldn’t even be worried if there were DNA binding proteins in the Mass Spec results. In the future, I think that a simple siRNA followed by a western or IP western would be the best way to obtain secondary characterization for a co-activator. However, since the right TF is detected in bands A-C, I think this is one is OK.
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford
EP400 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
exempt from standards
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line: K562, using the antibody A300-541A. The blot shows western blot analysis of input, flowthrough, immunoprecipitate and mock immunoprecipitate using IgG.Molecular Weight: 343.489
Submitter comment
We didn't have a suitable ladder in the lab for a protein of this size.
Reviewer comment
Ladder only goes to 250, so cannot evaluate a 343 KD molecule but we will give the submitter the benefit of the doubt.
Submitted by
Denis Salins
Lab
Michael Snyder, Stanford
EP400 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
exempt from standards
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line K562 using the antibody A300-541A. 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: 343.489.
Submitter comment
We analyzed all 4 bands by mass-spec and the target factor was detected.
Reviewer comment
Multiple immunoreactive bands, all were analyzed by IP-MS and target factor was found in bands A-C. Major band runs near expected size.
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford