Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (AB240647)

Immunohistochemical analysis of paraffin-embedded Human cardiac muscle tissue labeling HEXIM1 with ab240647 at 1/2000 dilution dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human cardiac muscle (PMID : 23300697). The section was incubated with ab240647 for 10 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (AB240647)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling HEXIM1 with ab240647 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (AB240647)

Immunofluorescent analysis of 100% methanol-fixed HeLa cells labelling HEXIM1 with ab240647 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (Green). Confocal image showing nuclear staining in HeLa cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (AB240647)

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling HEXIM1 with ab240647 at 1/2000 dilution dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human testis (PMID : 23300697). The section was incubated with ab240647 for 10 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins.

• ChIP

Supplier Data

ChIP - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (AB240647)

Chromatin was prepared from LNCaP cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 μg of chromatin, 5 μg of ab240647 (red), or 5 μg of rabbit normal IgG ab172730 (gray) and 20 μl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).

• IP

Unknown

Immunoprecipitation - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (AB240647)

HEXIM1 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) (treated with 10 μM MG-132 for 24 hours), whole cell lysate with ab240647 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab240647 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 10 μM MG-132 for 24 hours, whole cell lysate 10 ug

Lane 2 : ab240647 IP in HeLa treated with 10 μM MG-132 for 24 hours whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab240647 in HeLa treated with 10 μM MG-132 for 24 hours whole cell lysate

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (ab240647)

Predicted band size: 41 kDa

Observed band size: 60 kDa

false

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (AB240647)

Immunofluorescent analysis of 100% methanol-fixed MEF cells labelling HEXIM1 with ab240647 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 (Green). Confocal image showing nuclear staining in MEFs. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• ChIP

Supplier Data

ChIP - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (AB240647)

Chromatin was prepared from MEF cells according to the Abcam Dual-X-ChIP protocol. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 μg of chromatin, 5 μg of ab 240647 (red), or 5 μg of rabbit normal IgG ab172730 (gray) and 20 μl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are from paper PMCID : PMC4103662

• IP

Unknown

Immunoprecipitation - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (AB240647)

HEXIM1 was immunoprecipitated from 0.35 mg MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate with ab240647 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab240647 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate 10 ug

Lane 2 : ab240647 IP in MEF whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab240647 in MEF whole cell lysate

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

Exposure time : 3 minutes.

Lysate were made freshly and used in IP test immediately to minimize protein degradation. Incubation for immunoprecipitation was carried out overnight at 4°C.

All lanes:

Immunoprecipitation - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (ab240647)

Predicted band size: 41 kDa

Observed band size: 60 kDa

false

• WB

Lab

Western blot - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (AB240647)

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

The molecular weight observed is consistent with what has been described in the literature (PMID : 28254838)

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

Exposure time : 26 seconds.

All lanes:

Western blot - Anti-HEXIM1 antibody [EPR23430-12] - ChIP Grade (ab240647) at 1/1000 dilution

Lane 1:

LNCaP (human prostate carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

PC-12 (rat adrenal gland pheochromocytoma ) whole cell 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 1/100000 dilution

Predicted band size: 41 kDa

Observed band size: 60 kDa

false