Anti-HDAC2 antibody

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-HDAC2 antibody (AB7029)

HeLa cells were fixed with 4% formaldehyde in PEM buffer. The coverslip was incubated in blocking buffer of 5% powdered milk in TBS-T plus 0.02% sodium azide for 1 hour at room temperature. Blocking buffer was removed and primary antibody was added at a dilution of 1/600 and incubated overnight at 4 degrees celsius. The coverslips were then washed 4-5 times with blocking buffer for 5 minutes. Secondary antibody, goat anti-rabbit Alexa 594, was added at a dilution of 1/1000 and incubated at room temperature for one hour. From this point on coverslips were covered with foil to protect them from light. They were washed 5 times with TBS-T and then one time with PEM, for 5 minutes each wash. The coverslips were fixed 10-30 minutes in 4% formaldehyde in PEM buffer, then washed 3 times with PEM buffer for 5 minutes. 0.1M ammonium chloride in PEM buffer was added for 10 minutes to quench auto-florescence, and then slips were washed 2 times for 5 minutes in PEM followed by 3 washes for 5 minute

This image is courtesy of Michael Mancini, Baylor College of Medicine

• IP

Unknown

Immunoprecipitation - Anti-HDAC2 antibody (AB7029)

HDAC2 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to HDAC2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab7029.
Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band : Band : 60ka : HDAC2; 55kDa; Heavy Chain.

All lanes:

Immunoprecipitation - Anti-HDAC2 antibody (ab7029)

Predicted band size: 55 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-HDAC2 antibody (AB7029)

Immunoprecipitation analysis of HeLa whole cell lysates, ab7029 was used to precipitate HDAC3 from solution.

Lane 1:

Immunoprecipitation - Anti-HDAC2 antibody (ab7029) at 5 µL

Lane 2:

Immunoprecipitation - Anti-HDAC2 antibody (ab7029) at 10 µL

Lane 3:

Negative Control

All lanes:

HeLa whole cell lysate.

Predicted band size: 55 kDa

false

• WB

CiteAb

Western blot - Anti-HDAC2 antibody (AB7029)

Western Blotting using Anti-HDAC2 antibody, ab7029. Publication image from Lam, E. W. et al., 2006, Mol Cancer, 17018141. Legend direct from paper.

(A). TSA induces Akt and GSK3β dephosphorylation in MCF-7 breast cancer cells. MCF-7 cells were incubated with 1 µM TSA for the indicated times. Following incubation, the cells were harvested and lysates were resolved by SDS-PAGE. Proteins were detected using the indicated antibodies. (B). The relative amounts of pAkt and pGSK3β in A were measured by densitometry and normalised to the amount of p38/SAPK. Result is representative of at least three separate experiments. (C). Knockdown of class I HDAC proteins induces Akt and GSK3β dephosphorylation. MCF-7 cells were transfected with oligo pools specifically targeting HDAC1, 2, 3 or a non-targeting siRNA pool (NSC). 72 h after transfection, cells were harvested and lysed. Lysates were treated as in A and probed with the indicated antibodies. (D). siRNA-mediated GSK3β knockdown attenuates the cytotoxic effect of TSA on MCF-7 cells. MCF-7 cells were transfected with oligo pools specifically targeting GSK3β or a non-targeting siRNA pool (NSC). 24 h after transfection cells were harvested and reseeded in 96-well plates and incubated for 24 h. Cells were then treated with 1 µM TSA for 48 h and relative cell survival was measured as described in materials and methods. Results represent the mean ± S.E. from at least three separate experiments. * P < 0.001 TSA treated vs. untreated NSC siRNA cells, ** P < 0.001 TSA treated NSC vs. TSA treated GSK3β siRNA cells. Inset : Lysates from cells transfected in parallel were probed with antibodies directed against GSK3 to monitor siRNA efficiency. (E). Effect of GSK3β siRNA on TSA induced cytotoxicity. Cells were treated as in D and examined by flow cytometry (see materials and methods section). Result is representative of at least three separate experiments. (F). Effect of GSK3β inhibition on TSA induced cytotoxicity. MCF-7 cells were cultured in 96-well plates with 10-9 – 10-5 M TSA alone or in combination with 10 mM LiCl. Relative cell survival was determined after 48 h as described in materials and methods section. Result is representative of three separate experiments.

false