Anti-BTK (phospho Y551) antibody [EP267Y] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BTK (phospho Y551) antibody [EP267Y] - BSA and Azide free (AB247285)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Ramos (human Burkitt's lymphoma B lymphocyte) cells labelling BTK with primary antibody anti- BTK (phospho Y551) (ab40770) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150081) secondary antibody at 1/1000 dilution (2.0 μg/mL). Confocal image showing increased cytoplasmic staining in Ramos cells treated with pervanadate (1 mM) for 30 min and then the signal decreased after Alkaline Phosphatase treatment 37℃ for 1 hour. Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution (2.5 μg/mL). The nuclear counter stain is DAPI (blue).

The secondary antibody only control is : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution (2.0 μg/mL).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40770).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-BTK (phospho Y551) antibody [EP267Y] - BSA and Azide free (AB247285)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40770).

Flow cytometric analysis of 4% paraformaldehyde fixed, 0.1% Tween-20 permeabilized Untreated control (Left) / Ramos cell treated with 1mM pervanadate for 30min (Middle) / Ramos cell treated with 1mM pervanadate for 30min, then incubated with Alkaline phosphatase overnight (Right) cells labelling BTK (phospho Y551) with ab40770 at 1/2000 dilution (0.1µg) followed by a Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at a 1/5000 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

• WB

Lab

Western blot - Anti-BTK (phospho Y551) antibody [EP267Y] - BSA and Azide free (AB247285)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM/TBST This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40770).

Lanes 1 - 3:

Western blot - Anti-BTK (phospho Y551) antibody [EP267Y] - BSA and Azide free (ab247285) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-BTK (phospho Y551) antibody [EP267Y] (<a href='/en-us/products/primary-antibodies/btk-phospho-y551-antibody-ep267y-ab40770'>ab40770</a>) at 1/1000 dilution

Lane 1:

Untreated K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 15 µg

Lane 2:

K-562 treated with 1mM pervanadate for 30 min whole cell lysate at 15 µg

Lane 3:

K-562 treated with 1mM pervanadate for 30 min whole cell lysate, then the membrane treated with Alkaline Phosphatase for 1 hour at 15 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 1/20000 dilution

Predicted band size: 76 kDa

Observed band size: 75 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-BTK (phospho Y551) antibody [EP267Y] - BSA and Azide free (AB247285)

Blocking buffer and concentration : 5% NFDM/TBST Diluting buffer and concentration : 5% NFDM/TBST This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40770).

Lanes 1 - 3:

Western blot - Anti-BTK (phospho Y551) antibody [EP267Y] - BSA and Azide free (ab247285) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-BTK (phospho Y551) antibody [EP267Y] (<a href='/en-us/products/primary-antibodies/btk-phospho-y551-antibody-ep267y-ab40770'>ab40770</a>) at 1/1000 dilution

Lane 1:

Untreated Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate at 15 µg

Lane 2:

Ramos treated with 1mM pervanadate for 30 min whole cell lysate at 15 µg

Lane 3:

Ramos treated with 1mM pervanadate for 30 min whole cell lysate, then the membrane treated with Alkaline Phosphatase for 1 hour at 15 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 1/20000 dilution

Predicted band size: 76 kDa

Observed band size: 75 kDa

false

Exposure time: 7s

• Dot

Lab

Dot Blot - Anti-BTK (phospho Y551) antibody [EP267Y] - BSA and Azide free (AB247285)

Dot blot analysis of Human BTK non-phospho peptide (lane 1), Human BTK Y551 phospho peptide (lane 2), Human BMX non-phospho peptide (lane 3), Human BMX Y566 phospho peptide (lane 4), Human ITK non-phospho peptide (lane 5), Human ITK Y512 phospho peptide (lane 6), Human TEC non-phospho peptide (lane 7), Human TEC Y519 phospho peptide (lane 8), Human TXK non-phospho peptide (lane 9) and Human TXK Y420 phospho peptide (lane 10) with ab40770 at a dilution of 1/1000. ab97051 Peroxidase conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody at a dilution of 1/20,000.

Blocking buffer and concentration : 5% NFDM/TBST

Diluting buffer and concentration : 5% NFDM/TBST

Exposure time : 180 seconds

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40770).

• I-ELISA

Lab

Indirect ELISA - Anti-BTK (phospho Y551) antibody [EP267Y] - BSA and Azide free (AB247285)

ELISA using ab40770 at varying antibody concentrations and antigen concentration of 1000 ng/ml. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody. Substrate solution was p-nitrophenyl phosphate (PNPP). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab40770).