All lanes : Anti-CstF-64 antibody [EPR15698] (ab200837) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CSTF2 knockout HeLa cell lysate
Lane 3 : K-562 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 61 kDa
Observed band size: 65 kDa why is the actual band size different from the predicted?

Lanes 1-3: Merged signal (red and green). Green - ab200837 observed at 65 kDa. Red - loading control ab8245 observed at 36 kDa.

ab200837 Anti-CstF-64 antibody [EPR15698] was shown to specifically react with CstF-64 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265207 (knockout cell lysate ab257402) was used. Wild-type and CstF-64 knockout samples were subjected to SDS-PAGE. ab200837 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

All lanes : Anti-CstF-64 antibody [EPR15698] (ab200837) at 1/5000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate
Lane 2 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate
Lane 3 : THP-1 (Human monocytic leukemia cells) whole cell lysate
Lane 4 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate

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: 61 kDa
Observed band size: 61 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Flow Cytometry analysis of Jurkat (human acute T cell leukemia) labelling CstF-64 with purified ab200837 at 1/25000 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Alexa Fluor® 488 goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

 

Anti-CstF-64 antibody [EPR15698] (ab200837) at 1/200 dilution + Human testis tissue lysate

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 61 kDa
Observed band size: 61 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-CstF-64 antibody [EPR15698] (ab200837) at 1/5000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate with CSTF2 peptide
Lane 3 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate with CSTFT peptide

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

Predicted band size: 61 kDa
Observed band size: 61 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Based on sequence analysis, ab200837 shares 78% homology with family member CSTFT. The levels of XR were tested in the accompanying peptide blocking experiment.

Immunohistochemical analysis of paraffin-embedded Human cervix carcinoma tissue labeling CstF-64 with ab200837 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on Human cervix carcinoma tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only control: Used 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 Human tonsil tissue labeling CstF-64 with ab200837 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody at 1/500 dilution. Nuclear staining on Human tonsil tissue is observed. Counter stained with Hematoxylin.

Secondary antibody only: Used 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling CstF-64 with ab200837 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Nuclear staining on HeLa cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows;
-ve control 1: ab200837 at 1/500 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling CstF-64 with ab200837 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Nuclear staining on Jurkat cell line is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows;
-ve control 1: ab200837 at 1/500 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.

CstF-64 was immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate with ab200837 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab200837 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: K562 whole cell lysate10 µg (Input). Lane 2: ab200837 IP in K562 whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab200837 in K562 whole cell lysate.
Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 5 seconds