Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade

• IP

Supplier Data

Immunoprecipitation - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

HMGA1 was immunoprecipitated from 0.35mg of EL4 (mouse lymphoma T lymphocyte) whole cell lysate with ab252930 at 1/30 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

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

Lane 1:

EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 10 µg

Lane 2:

ab252930 IP in EL4 (mouse lymphoma T lymphocyte) whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab252930 in EL4 (human epidermoid carcinoma) whole cell lysate at 10 µg

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 17 kDa

Observed band size: 12 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

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).

All lanes:

Western blot - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (ab252930) at 1/1000 dilution

Lane 1:

HT-1080 (human fibrosarcoma epithelial cell), whole cell lysate at 20 µg

Lane 2:

NCCIT (human pluripotent embryonic carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

HCT116 (human colorectal carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

Human colon cancer tissue lysate at 20 µg

Lane 5:

Human testis tissue lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 12 kDa

Observed band size: 17 kDa

false

Exposure time: 136s

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

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).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

Intracellular 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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

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).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

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).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

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.

• IP

Unknown

Immunoprecipitation - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

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.

All lanes:

Immunoprecipitation - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (ab252930)

Predicted band size: 12 kDa

Observed band size: 17 kDa

false

• ChIP

Supplier Data

ChIP - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

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.

• WB

Lab

Western blot - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

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

Lanes 1 - 3:

Western blot - Anti-HMGA1 antibody [EPR22421-101] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/hmga1-antibody-epr22421-101-bsa-and-azide-free-ab255277'>ab255277</a>) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (ab252930) at 1/1000 dilution

Lane 1:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) nuclear fraction lysate at 15 µg

Lane 2:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) cytoplasm fraction lysate at 15 µg

Lane 3:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) membrane fraction lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 12 kDa

Observed band size: 17 kDa

false

Exposure time: 10s

• WB

Lab

Western blot - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (AB252930)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM/TBST We recommend using 1% SDS Hot lysis prepare method to get desired Western Blot results.

Lanes 1 - 4:

Western blot - Anti-HMGA1 antibody [EPR22421-101] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/hmga1-antibody-epr22421-101-bsa-and-azide-free-ab255277'>ab255277</a>) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-HMGA1 antibody [EPR22421-101] - ChIP Grade (ab252930) at 1/1000 dilution

Lane 1:

HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysate prepared in RIPA lysis method at 20 µg

Lane 2:

HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysate prepared in 1% SDS Hot lysis method at 20 µg

Lane 3:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate prepared in RIPA lysis method at 20 µg

Lane 4:

MDA-MB-231 (Human breast adenocarcinoma epithelial cell) whole cell lysate prepared in 1% SDS Hot lysis method at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 12 kDa

Observed band size: 17 kDa

false

Exposure time: 40s