Western blot - HDAC3 antibody (ab16047)

All lanes : Anti-HDAC3 antibody (ab16047) at 1 µg/ml

Lane 1 : Mouse 3T3 cell lysate
Lane 2 : Rat liver cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 50 kDa
Observed band size : 49 kDa (why is the actual band size different from the predicted?)


Exposure time : 30 seconds

ab16047 cross-reacts with Mouse 3T3 cell lysate but shows no cross-reactivity against Rat liver lysate.

Western blot

All lanes : Anti-HDAC3 antibody (ab16047) at 1 µg/ml

Lane 1 : HeLa nuclear lysate
Lane 2 : HeLa whole cell lysate
Lane 3 : A431 cell lysate
Lane 4 : Jurkat cell lysate
Lane 5 : HEK293 cell lysate
Lane 6 : HeLa nuclear lysate with HDAC3 peptide (ab16279) at 1 µg/ml
Lane 7 : HeLa whole cell lysate with HDAC3 peptide (ab16279) at 1 µg/ml
Lane 8 : A431 cell lysate with HDAC3 peptide (ab16279) at 1 µg/ml
Lane 9 : Jurkat cell lysate with HDAC3 peptide (ab16279) at 1 µg/ml
Lane 10 : HEK293 cell lysate with HDAC3 peptide (ab16279) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 50 kDa
Observed band size : 49 kDa (why is the actual band size different from the predicted?)


Exposure time : 30 seconds

Immunoprecipitation - Anti-HDAC3 antibody (ab16047)

HDAC3 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to HDAC3 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 ab16047.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 49kDa: HDAC3.

Immunocytochemistry - HDAC3 antibody (ab16047)

SK-N-SH cells were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100 and incubated for 1 hour with ab16047 (1/100). The antibody clearly stained the nucleus (red). The cells were counterstained with DAPI (blue). 100x magnification.

Darin McDonald, Cross Cancer Institute

Immunocytochemistry - HDAC3 antibody (ab16047)

Interphase HeLa cells incubated with ab16047 (1/500). The antibody shows predominantly nuclear staining. The ab16047 staining is shown in green. The cells were counterstained with DAPI (red).

Kirk McManus, University of British Columbia

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HDAC3 antibody (ab16047)

ab16047 staining HDAC3 in human breast cancer tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue underwent fixation in formaldehyde, heat mediated antigen retrieval in Citrate buffer pH 6.0 and blocking (5 minutes/peroxidase block then 10 minutes/protein block) for 15 minutes at 20°C. The primary antibody was diluted, 1/2000 and incubated with sample for 45 minutes at 20°C. A HRP conjugated goat polyclonal to rabbit IgG was used undiluted as secondary.

This image is courtesy of an Abreview submitted by Antibody Solutions Ltd.

Western blot - Anti-HDAC3 antibody (ab16047)

All lanes : Anti-HDAC3 antibody (ab16047) at 1 µg/ml

Lane 1 : Marker
Lane 2 : Zebrafish brain homogenate (20ug)
Lane 3 : Zebrafish heart homogenate (20ug)
Lane 4 : Zebrafish liver homogenate (20ug)

Secondary
Donkey polyclonal to Goat IgG – H&L – Pre-Adsorbed (HRP) at 1/6000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 50 kDa
Observed band size : 48 kDa (why is the actual band size different from the predicted?)


Exposure time : 5 minutes