1. Antibodies
  2. Homo sapiens
  3. WRNIP1

ENCAB000AMJ

Antibody against Homo sapiens WRNIP1

Homo sapiens
K562
characterized to standards
Homo sapiens
any cell type or tissue
partially characterized
Status
released
Source (vendor)
Abcam
Product ID
ab4731
Lot ID
GR33648
Characterized targets
WRNIP1 (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Purification
affinity
Antigen description
Immunogen: (Synthetic peptide) SRESYDAPPTPSGAC conjugated to KLH, corresponding to amino acids 107-120 of Human WHIP.
Antigen sequence
SRESYDAPPTPSGAC

Characterizations

WRNIP1 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
not reviewed
Caption
Immunoprecipitation of WHIP from K562 cells using ab4731 antibody. Lane 1: input nuclear lysate, Lane 2: material immunoprecipitated with ab4731, Lane 3: material immunoprecipitated using control IgG. Bands A was excised from the gel and subjected to analysis by mass spectrometry." IP followed by masspectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using ab4731, 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 22 proteins identified in band A, although 7 of these are also present in a control immunoprecipitation and are thus likely to present due to non-specific association with the IP matrix. Of the specifically immunoprecipitated proteins, WHIP and Sun-domain protein 2 are the most abundant. However, the Sun domain protein 2 is thought to localize to inner nuclear memebrane and link the nucleo- and cytoskeleton and thus unlikely to interefer with ChIP-Seq analysis using ab4731 (Tzur YB, Wilson KL, Gruenbaum Y (Oct 2006). "SUN-domain proteins: 'Velcro' that links the nucleoskeleton to the cytoskeleton". Nat Rev Mol Cell Biol. 7 (10): 782-8. doi:10.1038/nrm2003). WHIP is the most abundant transcription factor in band A (20 peptides). Based on these observations, this band is likely due to the presence of immunoprecipitated WHIP and ab4731 meets the ENCODE standard for validation by this criterion.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
WRNIP1 (Homo sapiens)
Method: immunoblot
Attachment PDF Icon
not reviewed
Caption
Western blot of K562, GM12878, HelaS3 and HepG2 nuclear extracts using anti-WHIP (ab4731) detects a protein of the molecular weight protein of WHIP (72kD). This band was analyzed further by IP-Mass Spec.
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
WRNIP1 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
Attachment from submitters
compliant
Caption
Immunoprecipitation of WRNIP1 from K562 cells using ab4731. Lane 1: input nuclear lysate. Lane 2: material immunoprecipitated with ab4731. Lane 3: material immunoprecipitated using control IgG. Band A was excised from gel and subject to analysis by mass spectrometry.
Submitted by
Minyi Shi
Lab
Michael Snyder, Stanford
Download
 WHIP_final.jpg
WRNIP1 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
Attachment PDF Icon
compliant
Caption
IP followed by masspectrometry: Briefly, protein was immunoprecipitated from K562 whole cell lysates using ab4731, 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 22 proteins identified in band A, although 7 of these are also present in a control immunoprecipitation and are thus likely to present due to non-specific association with the IP matrix. Of the specifically immunoprecipitated proteins, WHIP and Sun-domain protein 2 are the most abundant. However, the Sun domain protein 2 is thought to localize to inner nuclear memebrane and link the nucleo- and cytoskeleton and thus unlikely to interefer with ChIP-Seq analysis using ab4731 (Tzur YB, Wilson KL, Gruenbaum Y (Oct 2006). "SUN-domain proteins: 'Velcro' that links the nucleoskeleton to the cytoskeleton". Nat Rev Mol Cell Biol. 7 (10): 782–8. doi:10.1038/nrm2003). WHIP is the most abundant transcription factor in band A (20 peptides).
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
Download
 WHIP_final.pdf