Anti-BTK antibody [EPR20445] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling BTK with ab208937 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Strong cytoplasmic staining on lymphoid nodule in human colon (PMID : 25433814). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling BTK with ab208937 at 1/4000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Strong cytoplasmic staining in mantle zone and weaker cytoplasmic staining in the germinal center of human tonsil (PMID : 25433814). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

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

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized Jurkat (human T cell leukemia cell line from peripheral blood) (left panel) and Ramos (human Burkitt's lymphoma cell line) (right panel) cell lines labeling BTK with ab208937 at 1/600 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

Negative control : Jurkat cell line (PMID : 24759210).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

Immunofluorescent analysis of 100% methanol-fixed Ramos (human Burkitt's lymphoma cell line) and Jurkat (human T cell leukemia cell line from peripheral blood) cells labeling BTK with ab208937 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing cytoplasmic staining on Ramos cell line.

Negative control : Jurkat cell line (PMID : 24759210).

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cell lines labeling BTK with ab208937 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) was used as the secondary antibody at 1/5000 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized NR8383 (rat alveolar macrophage) cell lines labeling BTK with ab208937 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue).

Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) was used as the secondary antibody at 1/5000 dilution.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) cells labeling BTK with ab208937 at 1/250 (8.6 ug/ml) dilution followed by ab150081, Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed secondary antibody at 1/1000 (2 ug/ml) dilution (green). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used as a counterstain at 1/200 (2.5 ug/ml) dilution.

Confocal image showing cytoplasmic staining in RAW 264.7 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• WB

Supplier Data

Western blot - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM/TBST Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

All lanes:

Western blot - Anti-BTK antibody [EPR20445] (<a href='/en-us/products/primary-antibodies/btk-antibody-epr20445-ab208937'>ab208937</a>) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia cell) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 3:

NR8383 (rat lung macrophage (alveolar)) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

Blocking buffer and concentration :  5% NFDM/TBST Diluting buffer and concentration :  5% NFDM/TBST Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

All lanes:

Western blot - Anti-BTK antibody [EPR20445] (<a href='/en-us/products/primary-antibodies/btk-antibody-epr20445-ab208937'>ab208937</a>) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

false

Exposure time: 15s

• IP

Unknown

Immunoprecipitation - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

BTK was immunoprecipitated from 0.35 mg of Ramos (human Burkitt's lymphoma cell line) whole cell lysate with ab208937 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab208937 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Ramos whole cell lysate 10 μg (Input).

Lane 2 : ab208937 IP in Ramos whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab208937 in Ramos whole cell lysate.

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

Exposure time : 3 seconds.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

All lanes:

Immunoprecipitation - Anti-BTK antibody [EPR20445] (<a href='/en-us/products/primary-antibodies/btk-antibody-epr20445-ab208937'>ab208937</a>)

Predicted band size: 76 kDa

Observed band size: 76 kDa

false

• WB

Lab

Western blot - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM/TBST Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

All lanes:

Western blot - Anti-BTK antibody [EPR20445] (<a href='/en-us/products/primary-antibodies/btk-antibody-epr20445-ab208937'>ab208937</a>) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia cell) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 3:

NR8383 (rat lung macrophage (alveolar)) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 76 kDa

Observed band size: 76 kDa

false

Exposure time: 37s

• WB

Lab

Western blot - Anti-BTK antibody [EPR20445] - BSA and Azide free (AB227812)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM/TBST Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab208937).

All lanes:

Western blot - Anti-BTK antibody [EPR20445] (<a href='/en-us/products/primary-antibodies/btk-antibody-epr20445-ab208937'>ab208937</a>) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

Exposure time: 15s