Anti-CstF-64 antibody [EPR15698]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CstF-64 antibody [EPR15698] (AB200837)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-CstF-64 antibody [EPR15698] (AB200837)

Intracellular Flow Cytometry analysis of Jurkat (human acute T cell leukemia) labelling CstF-64with 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CstF-64 antibody [EPR15698] (AB200837)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CstF-64 antibody [EPR15698] (AB200837)

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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CstF-64 antibody [EPR15698] (AB200837)

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.

• IP

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Immunoprecipitation - Anti-CstF-64 antibody [EPR15698] (AB200837)

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

All lanes:

Immunoprecipitation - Anti-CstF-64 antibody [EPR15698] (ab200837)

Predicted band size: 61 kDa

false

• WB

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Western blot - Anti-CstF-64 antibody [EPR15698] (AB200837)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CstF-64 antibody [EPR15698] (ab200837) at 1/200 dilution

All lanes:

Human testis tissue lysate

Secondary

All lanes:

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

false

Exposure time: 3min

• WB

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Western blot - Anti-CstF-64 antibody [EPR15698] (AB200837)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CstF-64 antibody [EPR15698] (ab200837) at 1/5000 dilution

Lane 1:

K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate at 10 µg

Lane 2:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate at 10 µg

Lane 3:

THP-1 (Human monocytic leukemia cells) whole cell lysate at 10 µg

Lane 4:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg

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

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-CstF-64 antibody [EPR15698] (AB200837)

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:

Western blot - Anti-CstF-64 antibody [EPR15698] (ab200837) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CSTF2 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CSTF2 (CstF-64) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cstf2-cstf-64-knockout-hela-cell-line-ab265207'>ab265207</a>)

Lane 3:

K-562 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 61 kDa

Observed band size: 65 kDa

false

• WB

Supplier Data

Western blot - Anti-CstF-64 antibody [EPR15698] (AB200837)

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.

All lanes:

Western blot - 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

false