ENCAB000AIT

Antibody against Homo sapiens MXI1, Mus musculus MXI1

Homo sapiens
K562
characterized to standards
Homo sapiens
HepG2, HeLa-S3, neural cell, GM12878
characterized to standards with exemption
Mus musculus
any cell type or tissue
partially characterized
Homo sapiens
any cell type or tissue
Status
released
Source (vendor)
RD Systems
Product ID
AF4185
Lot ID
ZIJ0107031
Host
goat
Clonality
polyclonal
Purification
affinity
Isotype
IgG
Antigen description
Raised against E. coli-derived rhMxi1.

Characterizations

MXI1 (Homo sapiens)
neural cell
Method: immunoprecipitation
Attachment from submitter
exempt from standards
Caption
The ENCODE antibody standards document exempts some valuable and/or limiting samples from primary characterizations for well-characterized antibodies. They are given exemptions so that the samples can be conserved to carry out the downstream experiments.
Submitter comment
We do not have any of the H1-derived neurons that were distributed to the consortium for analysis in ENCODE2 left.
Reviewer comment
This cell type was exempted from primary characterization of antibody ENCAB000AIT by the ENCODE antibody review panel on October 17, 2016
Submitted by
Jessika Adrian
Lab
Michael Snyder, Stanford
MXI1 (Homo sapiens)
Method: immunoprecipitation followed by mass spectrometry
not reviewed
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
MXI1 (Homo sapiens)
Method: immunoprecipitation
not reviewed
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
MXI1 (Homo sapiens)
K562
Method: immunoprecipitation
Attachment from submitter
compliant
Caption
Immunoprecipitation of MXI from K562 cells using AF4185. Lane 1: input nuclear lysate, Lane 2: material immunoprecipitated with ab87525, Lane 3: material immunoprecipitated using control IgG. Band A was excised from the gel and subject to analysis by mass spectrometry. Expected size: 26kD
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
MXI1 (Mus musculus)
Method: immunoprecipitation followed by mass spectrometry
not reviewed
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
MXI1 (Mus musculus)
Method: immunoprecipitation
not reviewed
Submitted by
Michael Snyder
Lab
Michael Snyder, Stanford
MXI1 (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 AF4185, 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 trysinized 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).
Submitted by
Kathrina Onate
Lab
Michael Snyder, Stanford
MXI1 (Homo sapiens)
HeLa-S3K562GM12878HepG2
Method: immunoblot
Attachment from submitter
exempt from standards
Caption
Western blot analysis of nuclear lysates prepared from multiple cells lines loaded in the order: HeLa-S3,K562,GM12878,HepG2 using the antibody AF4185. Molecular Weight: 26.062.
Submitter comment
We observed one major band of ~40kD in all cell lines assayed, along with bands of lesser intensity at ~50kD and ~32kD. The major band migrates somewhat slower than predicted for MXI1 (~26kD), but a similar sized band in the K562 IP was confirmed to contain MXI1 by mass spectrometry.
Reviewer comment
The major band detected is ~40kD and greater than the allowed 20% deviation from the expected size (~26kD) according to the standards but the secondary IP followed by mass spectrometry characterization in K562 showed that MXI1 is detected in the slower migrating band at that higher size, and likely the case for other cell lines as well.
Submitted by
Nathaniel Watson
Lab
Michael Snyder, Stanford