All lanes : Anti-Cdk9 antibody [EPR22956-37] - ChIP Grade (ab239364) at 1/1000 dilution

Lane 1 : 293T (human embryonic kidney epithelial cell), whole cell lysate at 10 µg
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 10 µg
Lane 3 : Mouse lung tissue lysate at 20 µg
Lane 4 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 10 µg
Lane 5 : Mouse brain tissue lysate 20 ug
Lane 6 : Rat lung tissue lysate at 20 µg
Lane 7 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 10 µg
Lane 8 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 10 µg
Lane 9 : C6 (rat glial tumor glial cell) whole cell lysate at 10 µg

Secondary
Lanes 1-5 & 7-9 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 6 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 43 kDa
Observed band size: 42,55 kDa why is the actual band size different from the predicted?

Blocking and Dilution Buffer and concentration: 5% NFDM/TBST

Exposure times.

Lanes 1-3: 36 seconds Lanes 4-5: 5.5 seconds Lane 6: 3 minutes Lanes 7-8: 15 seconds Lane 9: 48 seconds 

CDK9 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 ug with ab239364 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab239364 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 (mouse embryonic fibroblast), whole cell lysate 10 ug

Lane 2: ab239364 IP in NIH/3T3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab239364 in NIH/3T3 whole cell lysate

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

Exposure time: 6 seconds

 

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling CDK9 with ab239364 at 1/2000 dilution (0.30 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat liver (PMID: 9766517, 11282025). The section was incubated with ab239364 for 10 mins at RT. 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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

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 ab239364 (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 PMC4103662 (PMID: 23663783)
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling CDK9 with ab239364 at 1/2000 dilution (0.30 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse liver (PMID: 9766517, 11282025) The section was incubated with ab239364 for 10 mins at RT. 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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human pancreatic cancer tissue labeling CDK9 with ab239364 at 1/2000 dilution (0.30 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human pancreatic cancer (PMID: 28231737) The section was incubated with ab239364 for 10 mins at RT. 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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Human pancreas tissue labeling CDK9 with ab239364 at 1/2000 dilution (0.30 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human pancreas (PMID: 28231737) The section was incubated with ab239364 for 10 mins at RT. 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 Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling CDK9 with ab239364 at 1/100 (6 ug/ml) dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cell line is observed. 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 ab239364 anti-CDK9 ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling CDK9 with ab239364 at 1/100 (6 ug/ml) dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing nuclear staining in HeLa cell line is observed. 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 ab239364 anti-CDK9 ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 ug/ml) dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryo) cells labelling CDK9 with ab239364 at 1/600 (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.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma) cells labelling CDK9 with ab239364 at 1/600 (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.

Chromatin was prepared from HeLa 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 ab239364 (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: PMC2756882.

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol