Chromatin was prepared from EL4 cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min.

The ChIP was performed with 25 µg of chromatin, 5 µg of ab252930 (red) or 5 μg of rabbit normal IgG (gray) was added to 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (sybr green approach).

Primers and probes are located in the first kb of the transcribed region.

All lanes : Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (ab252930) at 1/1000 dilution

Lane 1 : HT-1080 (human fibrosarcoma epithelial cell), whole cell lysate
Lane 2 : NCCIT (human pluripotent embryonic carcinoma epithelial cell), whole cell lysate
Lane 3 : HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate
Lane 4 : Human colon cancer tissue lysate
Lane 5 : Human testis tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 12 kDa
Observed band size: 17 kDa why is the actual band size different from the predicted?


Exposure time: 136 seconds

Blocking buffer and concentration 5% NFDM/TBST
Diluting buffer and concentration 5% NFDM/TBST

The 17-kDa band observed is consistent with what has been described in the literature
(PMID:27045138).

Low expression of HMGA1 is expected in the testis (lane 5).

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling HMGA1 with ab252930 at 1/5000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in germ cells of human testis (PMID:17935122) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

HMGA1 was immunoprecipitated from 0.35 mg HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysate with ab252930 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab252930 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/5000 dilution.

Lane 1: HCT 116 whole cell lysate 10 µg (Input).
Lane 2: ab252930 IP in HCT 116 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab252930 in HCT 116 whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 40 seconds.

Immunohistochemical analysis of paraffin-embedded human pancreas cancer tissue labeling HMGA1 with ab252930 at 1/5000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in human pancreas cancer (PMID: 18473350) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NCCIT (human pluripotent embryonic carcinoma epithelial cell) cells labeling HMGA1 with ab252930 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining in NCCIT cell line. Low expression cell line: MCF7（PMID: 12640109). The nuclear counter stain is DAPI (blue). Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).

The  negative control is the secondary antibody only.

Immunohistochemical analysis of paraffin-embedded human lung cancer tissue labeling HMGA1 with ab252930 at 1/5000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in human lung cancer (PMID:25344216) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized MCF7 (Human breast adenocarcinoma epithelial cell, Left) / NCCIT (Human pluripotent embryonic carcinoma epithelial cell, Right) cell lines labeling HMGA1 with ab252930 at 1/600 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody. Weak expression control: MCF7 (PMID:12640109).