ENCAB000AFA

Antibody against Homo sapiens CUX1

Homo sapiens
K562
characterized to standards
Homo sapiens
MCF-7
characterized to standards with exemption
Homo sapiens
HepG2, any cell type or tissue, SK-N-SH
partially characterized
Status
released
Source (vendor)
Santa Cruz Biotech
Product ID
sc-6327
Lot ID
E0709
Characterized targets
CUX1 (Homo sapiens)
Host
goat
Clonality
polyclonal
Purification
affinity
Antigen description
Raised against a peptide mapping at the C-terminus of CDP of mouse origin.

Characterizations

CUX1 (Homo sapiens)
Method: immunoprecipitation
Attachment from submitter
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line: HepG2, using the antibody sc-6327. The blot shows western blot analysis of input, flowthrough, immunoprecipitate and mock immunoprecipitate using IgG.Molecular Weight: 180.0
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford
CUX1 (Homo sapiens)
MCF-7
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line: MCF-7, using the antibody sc-6327. The blot shows western blot analysis of input, flowthrough, immunoprecipitate and mock immunoprecipitate using IgG.Molecular Weight: 180.0
Submitter comment
Multiple bands appear because Uniprot: [P39880] states that this gene has 11 isoforms. Mass of Isoforms 1-3 (Uniprot: [P39880-1]; [P39880-2]; [P39880-3]) are between 161-165kDa. Isoform 7 (Uniprot: [P39880-6]) is 147kDa. Isoform 4 and 8 (Uniprot: [Q13948-1] and [Q13948-2]) are about 77kDa. Isoform 10 (Uniprot: [Q13948-10]) is 75.6KDa. Isoforms 9 and 11 (Uniprot: [Q13948-9] and [P39880-9]) are 72kDa.
Reviewer comment
Multiple strong bands detected at sizes not within the expected size range.
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford
CUX1 (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 SC6327, 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 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). We report 29 proteins identified in band A. Of the specifically immunoprecipitated proteins, CDPis the most abundant (373 peptides).
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
CUX1 (Homo sapiens)
Method: motif enrichment
compliant
Caption
The motif for target CUX1 is represented by the attached position weight matrix (PWM) derived from ENCFF451PUN. Motif enrichment analysis was done by Dr. Zhizhuo Zhang (Broad Institute, Kellis Lab) using known motifs (http://compbio.mit.edu/encode-motifs/) and previously published ChIP-seq data (http://www.broadinstitute.org/~zzhang/motifpipeline/data/TrainSetInfo.txt). The accept probability score of the given transcription factor was calculated using a Bayesian approach. This analysis also includes three motif enrichment scores, computed by overlapping the motif instances with the given ChIP-seq peak locations, as well as an enrichment rank. For more information on the underlying statistical methods, please see the attached document. Accept probability score: 0.987908705 Global enrichment Z-score: 5.025478757 Positional bias Z-score: 8.118934406 Peak rank bias Z-score: 4.329485696 Enrichment rank: 45.0
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
CUX1 (Homo sapiens)
MCF-7
Method: immunoprecipitation
Attachment from submitter
not compliant
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line: MCF-7, using the antibody sc-6327. The blot shows western blot analysis of input, flowthrough, immunoprecipitate and mock immunoprecipitate using IgG.
Reviewer comment
Too difficult to determine whether faint shadow near expected size is in fact the protein of interest.
Submitted by
Denis Salins
Lab
Michael Snyder, Stanford
Download
CUX1 (Homo sapiens)
Method: immunoprecipitation
not reviewed
Caption
Immunoprecipitation was performed on nuclear lysates from K562 cells using antibody SC6327 against CDP. Lane1: Nuclear lysate. Lane 2: Unbound material from immunoprecipitation with SC6327. Lane 3: Bound material from immunoprecipitation with SC6327. Lane 4: Bound material from control immunoprecipitation with rabbit IgG. Arrow indicates band of expected size (180 kD) that is highly enriched in the specifically immunoprecipitated fraction. !!Immunoprecipitation from K562 nuclear lysate enriches a protein of ~180KD. There are some low molecular weight bands which could be possible degradation products. The other non specific bands are also present in control IP. Based on these observations, this antibody meets this ENCODE criterion.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
CUX1 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
not reviewed
Caption
Immunoprecipitation of CDP from K562 cells using SC6327. Lane 1: input nuclear lysate, Lane 2: material immunoprecipitated with SC6327, Lane 3: material immunoprecipitated using control IgG. Bands A was excised from the gel and subject to analysis by mass spectrometry.!!IP followed by mass spectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using SC6327, 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).! We report 29 proteins identified in band A. Of the specifically immunoprecipitated proteins, CDPis the most abundant (373 peptides).! !Based on these observations, this band is likely due to the presence of immunoprecipitated CDPand SC6327 meets the ENCODE standard for validation by this criterion.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
CUX1 (Homo sapiens)
HepG2
Method: immunoprecipitation
Attachment from submitter
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line: HepG2 using the antibody sc-6327. The image shows western blot analysis of input, flowthrough, immunoprecipitate, and mock immunoprecipitate using IgG. Target molecular weight: 165.0.
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford
CUX1 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
Immunoprecipitation was performed on nuclear lysates from K562 cells using antibody SC6327 against CDP. Lane1: Nuclear lysate. Lane 2: Unbound material from immunoprecipitation with SC6327. Lane 3: Bound material from immunoprecipitation with SC6327. Lane 4: Bound material from control immunoprecipitation with rabbit IgG. Arrow indicates band of expected size (180 kD) that is highly enriched in the specifically immunoprecipitated fraction. Comment: Immunoprecipitation from K562 nuclear lysate enriches a protein of ~180KD. There are some low molecular weight bands which could be possible degradation products. The other non specific bands are also present in control IP.
Submitter comment
Multiple bands appear because Uniprot: [P39880] states that this gene has 11 isoforms. Mass of Isoforms 1-3 (Uniprot: [P39880-1]; [P39880-2]; [P39880-3]) are between 161-165kDa. Isoform 7 (Uniprot: [P39880-6]) is 147kDa. Isoform 4 and 8 (Uniprot: [Q13948-1] and [Q13948-2]) are about 77kDa. Isoform 10 (Uniprot: [Q13948-10]) is 75.6KDa. Isoforms 9 and 11 (Uniprot: [Q13948-9] and [P39880-9]) are 72kDa.
Reviewer comment
Multiple strong bands detected at sizes not within the expected size range.
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
CUX1 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
compliant
Caption
Immunoprecipitation of CDP from K562 cells using SC6327. Lane 1: input nuclear lysate, Lane 2: material immunoprecipitated with SC6327, Lane 3: material immunoprecipitated using control IgG. Bands A was excised from the gel and subject to analysis by mass spectrometry.
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
CUX1 (Homo sapiens)
SK-N-SH
Method: immunoprecipitation
Attachment from submitter
Caption
Immunoprecipitation was performed on nuclear extracts from the cell line: SK-N-SH using the antibody sc-6327. The image shows western blot analysis of input, flowthrough, immunoprecipitate, and mock immunoprecipitate using IgG. Target molecular weight: 165.0.
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford