ENCAB000AFA
Antibody against Homo sapiens CUX1
Homo sapiens
K562
characterized to standards
Homo sapiens
MCF-7
characterized to standards with exemption
Homo sapiens
any cell type or tissue, HepG2, 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.
- External resources
Characterizations
CUX1 (Homo sapiens)
not submitted for review by lab
- 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
- Grant
- U54HG006996
- Download
- 1128_01_CDP_sc6327.jpg
CUX1 (Homo sapiens)
MCF-7
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
- Grant
- U54HG006996
- Download
- 1128_02_CDP_sc6327.jpg
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
- Grant
- U54HG004558
- Download
- CDP_final_AFA.pdf
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
- Grant
- U54HG006996
CUX1 (Homo sapiens)
MCF-7
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
- Grant
- U54HG006996
- Download
- Exp.812_CDP.jpg
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
- Grant
- U54HG004558
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
- Grant
- U54HG004558
CUX1 (Homo sapiens)
HepG2
not submitted for review by lab
- 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
- Grant
- U54HG006996
- Download
- IP 1149 HepG2 Cux1 sc6327.jpg
CUX1 (Homo sapiens)
K562
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
- Grant
- U54HG004558
- Download
- IP-K562 Snyder AFA.png
CUX1 (Homo sapiens)
K562
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
- Grant
- U54HG004558
- Download
- IP-MS_CUX1 Snyder AFA.png
CUX1 (Homo sapiens)
SK-N-SH
not submitted for review by lab
- 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
- Grant
- U54HG006996
- Download
- IP1148 SK-N-SH CUX1 sc6327x.jpg