Anti-CHD5 抗体 [EPR23858-3-8] (BSA and Azide free) (ab300438)

All lanes : Anti-CHD5 antibody [EPR23858-3-8] (ab300437) at 1/1000 dilution

Lane 1 : PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate
Lane 2 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : IMR-32 (human neuroblastoma neuroblast) whole cell lysate

Lysates/proteins at 40 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Observed band size: 260 kDa why is the actual band size different from the predicted?

This data was developed using ab300437, the same antibody clone in a different buffer formulation. 

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

The molecular weight observed is consistent with what has been described in the literature (PMID: 25247294).

Negative control: HeLa (PMID: 21931736).

Lysates were made freshly and used in WB immediately to minimize protein degradation.

This blot of lane 3 was developed using a high sensitivity ECL substrate.

Exposure time: Lanes 1-2: 3 minutes; lane 3:147 seconds.

This data was developed using ab300437, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling CHD5 with ab300437 at 1/2000 dilution (0.237 µg/mL) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: No staining on human spleen. The section was incubated with ab300437 at 4°C overnight. 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 using ab208572 (Universal HIER antigen retrieval reagent)

This data was developed using ab300437, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling CHD5 with ab300437 at 1/2000 dilution (0.237 µg/mL) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human cerebrum. The section was incubated with ab300437 at 4°C overnight. 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 using ab208572 (Universal HIER antigen retrieval reagent)

This data was developed using ab300437, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling CHD5 with ab300437 at 1/2000 dilution (0.237 µg/mL) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human testis. The section was incubated with ab300437 at 4°C overnight. 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 using ab208572 (Universal HIER antigen retrieval reagent)

This data was developed using ab300437, the same antibody clone in a different buffer formulation.

CHD5 was immunoprecipitated from 0.35 mg PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate with ab300437 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab300437 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.

Lane 1: PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate 10 µg

Lane 2: ab300437 IP in PANC-1 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300437 in PANC-1 whole cell lysate

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

This data was developed using ab300437, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PANC-1(human pancreatic epithelioid carcinoma epithelial cells) labeling CHD5 with ab300437 at 1/500 (0.948 µg/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 (2 µg/ml) dilution (Green). Confocal image showing nuclear staining in PANC-1 cell line. Negative control: HeLa (PMID: 29682202). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 (2.5 µg/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 (2 µg/ml) dilution.

This data was developed using ab300437, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell, Left) / PANC-1 (human pancreatic epithelioid carcinoma epithelial cell, Right) cells labelling CHD5 with ab300437 at 1/500 dilution (0.1µg) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Negative control: HeLa (PMID: 29682202).