Immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue labeling 53BP1 with ab21083 at 1/4000 dilution. Nuclear staining is observed. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

All lanes : Anti-53BP1 antibody (ab21083) at 1/500 dilution

Lane 1 : 53BP1 shRNA non-transfected HeLa whole cell extracts
Lane 2 : 53BP1 shRNA transfected HeLa whole cell extracts

Lysates/proteins at 50 µg per lane.

Secondary
All lanes : HRP-conjugated anti-rabbit IgG antibody

Predicted band size: 220 kDa
Observed band size: 350 kDa why is the actual band size different from the predicted?

5% SDS-PAGE

All lanes : Anti-53BP1 antibody (ab21083) at 1/2000 dilution

Lane 1 : 293T whole cell lysate/extract
Lane 2 : A431 whole cell lysate/extract
Lane 3 : HeLa whole cell lysate/extract
Lane 4 : HepG2 whole cell lysate/extract
Lane 5 : A375 whole cell lysate/extract

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : HRP-conjugated anti-rabbit IgG antibody

Predicted band size: 220 kDa
Observed band size: 350 kDa why is the actual band size different from the predicted?

5% SDS-PAGE.

Running conditions: 80V, 15min; 140V, 40min.

Transfer condition: Semi-dry, 18 V, 60min (Nitrocellulose membrane).

Blocking condition: 5% non-fat milk in TBST, RT, 60min.

Primary antibody incubation: 1/2000, 4?, overnight.

Secondary antibody incubation: Rabbit IgG antibody (HRP), 1/10,000, RT, 1hr. 

Washing condition: 5 ml TBST, 4 x 5min.

Exposure system: Trident plus Western HRP Substrate.

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labeling 53BP1 with ab21083 at 1/4000 dilution. Nuclear staining is observed. Antigen Retrieval: Citrate buffer, pH 6.0, 15 min.

All lanes : Anti-53BP1 antibody (ab21083) at 1/500 dilution

Lane 1 : Neuro2A whole cell extracts
Lane 2 : C8D30 whole cell extracts
Lane 3 : NIH-3T3 whole cell extracts

Lysates/proteins at 30 µg per lane.

Secondary
All lanes : HRP-conjugated anti-rabbit IgG antibody

Predicted band size: 220 kDa
Observed band size: 350 kDa why is the actual band size different from the predicted?

5% SDS-PAGE

ab21083 (2µg/ml) staining 53BP1 in human Brain: Cerebellum using an automated system (DAKO Autostainer Plus). Using this protocol there is ubiquitous nuclear staining throughout.
Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.