重组Anti-GATA1抗体[EPR17362] - ChIP Grade

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GATA1 antibody [EPR17362] - ChIP Grade (AB181544)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling GATA1 with ab181544 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on leukocyte of Human colon stroma is observed. Counterstained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-GATA1 antibody [EPR17362] - ChIP Grade (AB181544)

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling GATA1 with ab181544 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on leukocyte of Human cervical cancer is observed. Counterstained with Hematoxylin.

Negative control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ChIP

Supplier Data

ChIP - Anti-GATA1 antibody [EPR17362] - ChIP Grade (AB181544)

Chromatin was prepared from K562 (Human chronic myelogenous leukemia cells from bone marrow) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 5μg of ab181544 (blue), and 20μl of Anti rabbit IgG sepharose beads. 5μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

"pro" stands for promoter region, while "NC2" stands for negative control which is negative loci at the promoter region.

• IP

Supplier Data

Immunoprecipitation - Anti-GATA1 antibody [EPR17362] - ChIP Grade (AB181544)

GATA1 was immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell extract with ab181544 at 1/70 dilution. Western blot was performed from the immunoprecipitate using ab181544 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1 : K562 whole cell extract 10 μg (Input). Lane 2 : ab181544 IP in K562 whole cell extract. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab181544 in K562 whole cell extract.
Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-GATA1 antibody [EPR17362] - ChIP Grade (ab181544)

Predicted band size: 43 kDa

false

• WB

Lab

Western blot - Anti-GATA1 antibody [EPR17362] - ChIP Grade (AB181544)

False colour image of Western blot : Anti-GATA1 antibody [EPR17362] - ChIP Grade staining at 1/10000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab181544 was shown to bind specifically to GATA1. A band was observed at 48 kDa in wild-type K562 cell lysates with no signal observed at this size in GATA1 knockout cell line ab285360 (knockout cell lysate ab289686). To generate this image, wild-type and GATA1 knockout K562 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-GATA1 antibody [EPR17362] - ChIP Grade (ab181544) at 1/10000 dilution

Lane 1:

Wild-type K562 cell lysate at 20 µg

Lane 2:

GATA1 [C116] knockout K562 cell lysate at 20 µg

Lane 3:

MOLT-4 cell lysate

Lane 4:

HeLa cell lysate

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 43 kDa

Observed band size: 48 kDa

false

• WB

Lab

Western blot - Anti-GATA1 antibody [EPR17362] - ChIP Grade (AB181544)

Western blot : Anti-GATA1 antibody [EPR17362] (ab181544) staining at 1/10000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab181544 was shown to bind specifically to GATA1. A band was observed at 45 kDa in wild-type K562 cell lysates with no signal observed at this size in GATA1 knockout cell line ab285360 (knockout K562 cell lysate). To generate this image, wild-type and GATA1 knockout K562 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-GATA1 antibody [EPR17362] - ChIP Grade (ab181544) at 1/10000 dilution

Lane 1:

Wild-type K562 cell lysate at 20 µg

Lane 2:

GATA1 knockout K562 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 45 kDa

false

• WB

Supplier Data

Western blot - Anti-GATA1 antibody [EPR17362] - ChIP Grade (AB181544)

HeLa cells do not express GATA1. The expression profile observed in HeLa is consistent with the literature (PMID : 27010793, PMID : 17196618, PMID : 22235304, PMID : 20823267).

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-GATA1 antibody [EPR17362] - ChIP Grade (ab181544) at 1/10000 dilution

Lane 1:

K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate at 5 µg

Lane 2:

HEL (Human bone marrow erythroleukemia) whole cell lysate at 5 µg

Lane 3:

MOLT-4 (Human lymphoblastic leukemia cell line) whole cell lysate at 5 µg

Lane 4:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 5 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-GATA1 antibody [EPR17362] - ChIP Grade (AB181544)

False colour image of Western blot : Anti-GATA1 antibody [EPR17362] - ChIP Grade staining at 1/10000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab181544 was shown to bind specifically to GATA1. A band was observed at 47 kDa in wild-type K562 cell lysates with no signal observed at this size in GATA1 knockout cell line ab285360 (knockout cell lysate ab289686). To generate this image, wild-type and GATA1 knockout K562 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% milk in TBS-0.1% Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-GATA1 antibody [EPR17362] - ChIP Grade (ab181544) at 1/10000 dilution

Lane 1:

Wild-type K562 cell lysate

Lane 2:

GATA1 knockout K562 cell lysate at 20 µg

Lane 3:

HL-60 cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Observed band size: 47 kDa

false