Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A549 (human lung carcinoma epithelial cell) cells labeling BAF170 with ab243634 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in A549 cell line. The nuclear counter stain is DAPI (Blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (Red).

PBS only control: Used PBS instead of primary antibody, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution. 

All lanes : Anti-BAF170 antibody [EPR23066-21] (ab243634) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 2 : RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate
Lane 4 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 133 kDa
Observed band size: 170 kDa why is the actual band size different from the predicted?

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

This antibody also recognizes weak unidentifiable bands below 100kDa.

Exposure time: 3 seconds.

Immunohistochemical analysis of paraffin-embedded human stomach tissue labeling BAF170 with ab243634 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human stomach. The section was incubated with ab243634 for 15 mins at RT. The immunostaining staining 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.

Flow cytometric analysis of Neuro-2a (mouse neuroblastoma neuroblast) cells labelling BAF170 with ab243649 at 1/500  (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). Goat anti rabbit IgG (Alexa Fluor^® 647, ab150077) at 1/2000 dilution was used as the secondary antibody.

BAF170 was immunoprecipitated from 0.35 mg K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate with ab243634 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab243634 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10ug

Lane 2: ab243634 IP in K-562 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab243634 in K-562 whole cell lysate

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

Exposure time: 30 seconds.

 

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (mouse neuroblastoma neuroblast) cells labeling BAF170 with ab243634 at 1/100 dilution, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing nuclear staining in Neuro-2a cell line. The nuclear counter stain is DAPI (Blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (Red).

PBS only control: Used PBS instead of primary antibody, followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution. 

All lanes : Anti-BAF170 antibody [EPR23066-21] (ab243634) at 1/1000 dilution

Lane 1 : K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 20 µg
Lane 2 : A549 (human lung carcinoma epithelial cell), whole cell lysate at 20 µg
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg
Lane 4 : 293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg
Lane 5 : C2C12 (mouse myoblasts myoblast), whole cell lysate at 20 µg
Lane 6 : Mouse brain tissue lysate at 10 µg
Lane 7 : Mouse lung tissue lysate at 10 µg
Lane 8 : Rat brain tissue lysate at 10 µg
Lane 9 : Rat liver tissue lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 133 kDa
Observed band size: 170 kDa why is the actual band size different from the predicted?

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

This antibody also recognizes weak unidentifiable bands below 100kDa. 

Exposure time: 3 seconds.

Immunohistochemical analysis of paraffin-embedded rat hippocampus tissue labeling BAF170 with ab243634 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat hippocampus (PMID: 27392482). The section was incubated with ab243634 for 15 mins at RT. The immunostaining staining 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 mouse hippocampus tissue labeling BAF170 with ab243634 at 1/1000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse hippocampus (PMID: 27392482) The section was incubated with ab243634 for 15 mins at RT. The immunostaining staining 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.

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized A549 (human lung carcinoma epithelial cell) cells labelling BAF170 with ab243634 at 1/500 (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.