ENCAB125OWA

Alternate accession: ENCAB000AEL

Antibody against Homo sapiens BRCA1

Homo sapiens
K562, HeLa-S3
characterized to standards
Homo sapiens
any cell type or tissue
partially characterized
Homo sapiens
HepG2, GM12878
not characterized to standards
Status
released
Source (vendor)
Bethyl Labs
Product ID
A300-000A
Lot ID
2
Characterized targets
BRCA1 (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Aliases
bradley-bernstein:PchAb 844
External resources

Characterizations

BRCA1 (Homo sapiens)
Method: immunoprecipitation
Attachment from submitter
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line: A549, using the antibody A300-000A. The blot shows western blot analysis of input, flowthrough, immunoprecipitate and mock immunoprecipitate using IgG.Molecular Weight: 207.7
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford
BRCA1 (Homo sapiens)
Method: immunoblot
Attachment from submitter
Submitted by
Noam Shoresh
Lab
Bradley Bernstein, Broad
BRCA1 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
compliant
Caption
IP followed by mass spectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using A300-000A, 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). Based on these observations, this band is likely due to the presence of immunoprecipitated BRCA1 and A300-000A meets the ENCODE standard for validation by this criterion.
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
BRCA1 (Homo sapiens)
Method: immunoprecipitation
Attachment from submitter
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line: HEK293T, using the antibody A300-000A. The blot shows western blot analysis of input, flowthrough, immunoprecipitate and mock immunoprecipitate using IgG.Molecular Weight: 207.7
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford
BRCA1 (Homo sapiens)
HeLa-S3
Method: immunoblot
compliant
Caption
Western blots on nuclear lysates from cell lines GM12878 (Lane1), K562 (Lane2), HeLaS3 (Lane3), and HepG2 (Lane4). A band of ~207kD is detected by Western blotting with A300-000A in K562 and HelaS3 nuclear lysates
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
BRCA1 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
compliant
Caption
Immunoprecipitation of BRCA1 from K562 cells using A300-000A. Lane 1: input nuclear lysate, Lane 2: material immunoprecipitated with A300-000A, Lane 3: material immunoprecipitated using control IgG. Bands A was excised from the gel and subject to analysis by mass spectrometry. IP followed by masspectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using A300-000A, 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). P followed by masspectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using A300-000A, 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). Based on these observations, this band is likely due to the presence of immunoprecipitated BRCA1 and A300-000A meets the ENCODE standard for validation by this criterion.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
BRCA1 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
compliant
Caption
Immunoprecipitation of BRCA1 from K562 cells using A300-000A. Lane 1: input nuclear lysate, Lane 2: material immunoprecipitated with A300-000A, Lane 3: material immunoprecipitated using control IgG. Bands A was excised from the gel and subject to analysis by mass spectrometry. IP followed by masspectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using A300-000A, and the IP fraction was loaded on a 10% polyacrylamide gel (NuPAGE Bis-Tris Gel) and separated with an Invitrogen NuPAGE electrophoresis system.
Reviewer comment
No band visible in IP-western but the factor is detected in the cut band by mass-spec.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
BRCA1 (Homo sapiens)
GM12878K562HeLa-S3HepG2
Method: immunoblot
Attachment from submitter
not compliant
Caption
Western blots on nuclear lysates from cell lines GM12878 (Lane1), K562 (Lane2), HeLaS3 (Lane3), and HepG2 (Lane4). A band of ~207kD is detected by Western blotting with A300-000A in K562 and HelaS3 nuclear lysates
Reviewer comment
Major/marked band not within 20% of expected size
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford