ENCAB000AHN

Antibody against Homo sapiens HMGN3

Homo sapiens
at least one cell type or tissue
awaiting characterization
Status
released
Source (vendor)
Novus
Product ID
NB100-61076
Lot ID
001
Characterized targets
HMGN3 (Homo sapiens)
Host
rabbit
Clonality
polyclonal
Purification
Protein A/G
Antigen description
Raised against an amino acids 60 to 99 of human HMGN3.

Characterizations

HMGN3 (Homo sapiens)
Method: immunoblot
not reviewed
Caption
In an immunoblot probed with NB100-60411, we observe a major band consistent with the expected size of CHD1 (197kD) in lysates from cell lines K562 and HeLaS3. The band is also observed with lysates from GM12878 cells, but at low abundance. This band is specifically immunoprecipitated from K562 cells, as are several minor bands (likely degradation products) and each is identified by mass spectrometry as CHD1 (see second validation) Therefore, NB100-60411 meets this criterion for validation.
Submitted by
Kevin Struhl
Lab
Michael Snyder, Stanford
HMGN3 (Homo sapiens)
Method: immunoprecipitation
not reviewed
Caption
Immunoprecipitation of CHD1 from K562 cells using NB100-60411. Lane 1: input nuclear lysate, Lane 2: material immunoprecipitated with ab87525, Lane 3: material immunoprecipitated using control IgG. Band A, the most abundant band and consisent with the expected size of CHD1, along with minor bands B and C were excised from the gel and subject to analysis by mass spectrometry. IP followed by mass spectrometry: Briefly, K562 whole cell lysates were immunoprecipitated using NB100-60411, 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 20 proteins identified in band A, of which 15 are also identified in a control immunoprecipitation. CHD1 is by far the most abundant of the specifically immunoprecipitated proteins ( 38 peptides). CHD1 is also identified in minor bands B (8 peptides) and C (7 peptides) and is the only chromosome associated protein from either band that is specifically immunoprecipitated. Based on these observations, the major band is likely due to the presence of immunoprecipated CHD1 and NB100-60411 meets the ENCODE standard for validation by this criterion.
Submitted by
Kevin Struhl
Lab
Michael Snyder, Stanford