Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) cells labeling Bcl G/BCL2L14 with ab184925 at 1/250 dilution, followed by Goat anti-rabbit I Alexa Fluor® 488 (IgG) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic staining on MCF7 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (goat anti-mouse AlexaFluor®594 secondary) at 1/500 dilution (red).
The negative controls are as follows:
-ve control 1: ab184925 at 1/250 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

 

All lanes : Anti-Bcl G/BCL2L14 antibody [EPR17666] (ab184925) at 1/2000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 2 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysates
Lane 3 : A549 (Human lung carcinoma) whole cell lysates
Lane 4 : Human fetal liver lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 37 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-Bcl G/BCL2L14 antibody [EPR17666] (ab184925) at 1/5000 dilution + Human testis lysates at 10 µg

Secondary
Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 37 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Bcl G/BCL2L14 antibody [EPR17666] (ab184925) at 1/2000 dilution

Lane 1 : Mouse brain lysates
Lane 2 : Mouse heart lysates
Lane 3 : Mouse kidney lysates
Lane 4 : Mouse spleen lysates
Lane 5 : Rat brain lysates
Lane 6 : Rat heart lysates
Lane 7 : C6 (Rat glial tumor cells) whole cell lysates
Lane 8 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates
Lane 9 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 10 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 37 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Bcl G/BCL2L14 with ab184925 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasm staining on lymphocytes of Human tonsil is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

 

J Biol Chem. 2001 Jan 26;276(4):2780-5.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human squamous cell carcinoma of cervix tissue labeling Bcl G/BCL2L14 with ab184925 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic staining on tumor cells of Human cervical squamous cell carcinoma is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling Bcl G/BCL2L14 with ab184925 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic staining on mouse kidney tubules is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling Bcl G/BCL2L14 with ab184925 at 1/500 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary at 1/500 dilution. Cytoplasmic staining on glandular epithelium of rat colon is observed. Counter stained with Hematoxylin.

Negative control: Using PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometric analysis of 2% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling Bcl G/BCL2L14 with ab184925 at 1/130 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

Bcl G/BCL2L14 was immunoprecipitated from 1mg of HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell extract with ab184925 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab184925 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution. Lane 1: HeLa whole cell extract. Lane 2: Rabbit monoclonal IgG (ab172730) instead of ab184925 in HeLa whole cell extract.

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