ENCAB563QWH

Antibody against Homo sapiens RNF2

Homo sapiens
HepG2, K562, endothelial cell of umbilical vein, A549, GM12878
characterized to standards with exemption
Status
released
Source (vendor)
Cell Signaling
Product ID
5694BF
Lot ID
2
Characterized targets
RNF2 (Homo sapiens)
Host
rabbit
Clonality
monoclonal
Antigen description
ring finger protein 2
Aliases
bradley-bernstein:PchAb 1146
External resources

Characterizations

RNF2 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment from submitter
exempt from standards
Caption
This mass spec was performed for ENCAB000BEA. We have been assured that this lot is only different in batch of purification. As there is no more of this lot to characterize we are submitting the work for the other lot. IP followed by mass spectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using 5694S, 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 treated with trypsin using the in-gel digestion method. Digested proteins were analyzed on a LTQ-Orbitrap (Thermo Scientific) b 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).
Submitter comment
This antibody is monoclonal and a second lot, ENCAB000BEA, has a valid secondary.
Reviewer comment
This is a monoclonal antibody corresponding to lot 2 from Cell Signaling. Lot 1 was characterized to standards in another cell type. Because this is a monoclonal and the next lot number (which Cell Signaling has told me in the past means that they just did a second purification from a large batch), I think we could give this an exemption.
Submitted by
Nina Farrell
Lab
Bradley Bernstein, Broad
RNF2 (Homo sapiens)
K562HepG2GM12878endothelial cell of umbilical veinA549
Method: immunoblot
Attachment from submitter
compliant
Caption
Nuclear lysates from K562 (10ug), HepG2 (10 ug), GM12878 (10ug), and HUVEC (10ug) were loaded into a 4-12% Bis-Tris gel in 1X MOPS running buffer. After separation, the samples were transferred to a nitrocellulose membrane using iblot. Membrane was blocked for an hour in room temperature, with 5% BSA in TBS-T and blotted with primary antibody in the appropriate concentration over night at 4c. Membrane was washed and blotted with secondary HRP-conjugated antibody. Detection was made with Optiblot ECL Detect Kit (ab133406) for 2 min. A range of bands were detected at the expected size.
Submitter comment
The antibody review board exempted A549 data from primary characterization requirement based on the quality of the ChIP of the two replicates and the other cell lines 03-05-2018
Reviewer comment
Peggy: I would be supportive of releasing the A549 dataset.
Submitted by
Noam Shoresh
Lab
Bradley Bernstein, Broad