ENCAB961EZQ

Antibody against Homo sapiens PAF1

Homo sapiens
A549, K562, H1-hESC, HepG2, GM12878, endothelial cell of umbilical vein
not characterized to standards
Status
released
Source (vendor)
Bethyl Labs
Product ID
A300-173A
Lot ID
2
Characterized targets
PAF1 (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Antigen description
RNA polymerase II-associated factor 1 homolog (PAF1); Pancreatic differentiation protein 2 (PD2)
Aliases
bradley-bernstein:PchAb 250
External resources

Characterizations

PAF1 (Homo sapiens)
Method: ChIP-seq comparison
not compliant
Caption
This validation relies on the use of antibodies to a chromatin regulator and a functionally related histone modification in K562 cells, and the demonstration that highly similar patterns of enrichment are obtained with each antibody. The first track shown used an antibody to H3K36me3 previously characterized to Encode standards (PchAb 1151, ENCAB405MHV), and the second track shown used an antibody to PAF1 (PchAb 250, ENCAB961EZQ). The final track shows an input control track for reference.
Submitter comment
We realize that by initial inspection it does not meet typical compliance metrics, since the correlation score is unspecified. But we believe that the other information in the secondary may add information to contribute to your confidence in the antibody and the datasets. If not, we do not have any further information to provide and will support the resulting decision on these datasets.
Reviewer comment
There are a few places in the genome where PAF1 overlaps H3K36me3 (such as the one you used in the secondary validation document and the attached document called Comparison2- pool fold change is the hES PAF1 data). However, there are also a lot of places where the correlation is just not there (see the other attached document). I think that the overall correlation between PAF1 and H3K36me3 would be pretty bad on a genome-wide scale. We can’t know if this is because of antibody issues or perhaps PAF1 is only supposed to be at a small subset of genomic locations that have H3K36me3. This is why I would be reluctant to release this data. Is there any way to perform an siRNA knockdown, followed by a western blot to confirm the antibody?
Submitted by
Nina Farrell
Lab
Bradley Bernstein, Broad
PAF1 (Homo sapiens)
K562HepG2GM12878endothelial cell of umbilical vein
Method: immunoblot
Attachment from submitter
compliant
Caption
Nuclear lysates from K562(10ug), GM12878 (10ug),HUVEC (10ug), HepG2 (10ug), were loaded into a 4-12% Bis-Tris gel in 1X MESrunning 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 stronger band of expected size was detected (~75kDa).
Submitted by
Noam Shoresh
Lab
Bradley Bernstein, Broad
PAF1 (Homo sapiens)
A549H1-hESC
Method: immunoblot
Attachment from submitter
compliant
Caption
Nuclear lysates from A549 (10ug) and H1 (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. Bands were detected at the expected size.
Submitted by
Noam Shoresh
Lab
Bradley Bernstein, Broad