BD PhosflowTM PE Mouse anti-Btk (pY223)/Itk (pY180)

Description

Analyses of BTK pY223 / ITK pY180 expression by Human and Mouse Cells. Human Cells Panel 1a: Flow cytometric analysis of BTK pY223 expressed by human Ramos cells. Serum-starved Ramos cells Human Burkitts lymphoma cell line were either not stimulated dashed line histogram or stimulated solid line histogram with Goat F ab 2 Anti-Human IgM Southern Biotech, Cat. No. 2022-14 at 37C for 2 minutes. Cells were fixed in BD Cytofix Buffer Cat. No. 554655; 37C for 10 min and permeabilized in BD Phosflow Perm Buffer III Cat. No. 558050 on ice 30 min . Cells were then stained with BD Phosflow PE Mouse Anti-Btk pY223 /Itk pY180 Cat. No. 562753 . Histograms showing BTK pY223 expression were generated for gated events with the forward and side-light scatter characteristics of intact cells using a BD FACSCanto II Flow Cytometer System. Panel 1b: Western blot analysis of BTK pY223 expressed by Ramos cells. Lysates from 1 million unstimulated C and Anti-IgM stimulated T Ramos cells were blotted using Purified Btk pY223 /Itk pY180 antibody 0.125, 0.063, and 0.032 mug/ml for Lanes 1, 2, and 3, respectively , HRP Goat Anti-Mouse Ig Cat. No. 554002 and a chemiluminescent detection system. BTK pY223 was identified as band of 77 kDa. Mouse Cells Panel 2a: Flow cytometric analysis of BTK pY223 expressed by mouse A20 cells. A20 cells mouse B lymphoma cell line were either not stimulated dashed line histogram or were stimulated solid line histogram with Rabbit Anti-Mouse IgG H+L Jackson, Cat. No. 315-005-003 at 37 C for 2 minutes. The cells were fixed, permed, stained and analyzed by flow cytometry as described above. Panel 2b: Western blot analysis of BTK pY223 expressed by mouse A20 cells. Lysates from 1 million untimulated C and Anti-IgM stimulated T A20 cells were blotted as described above with Purified Mouse Anti-Btk pY223 /Itk pY180 antibody at 0.032 mug/ml. Lower MW band at 42 kDa represents actin. Analyses of BTK pY223 / ITK pY180 expression by Human and Mouse Cells. Human Cells Panel 1a: Flow cytometric analysis of BTK pY223 expressed by human Ramos cells. Serum-starved Ramos cells Human Burkitts lymphoma cell line were either not stimulated dashed line histogram or stimulated solid line histogram with Goat F ab 2 Anti-Human IgM Southern Biotech, Cat. No. 2022-14 at 37C for 2 minutes. Cells were fixed in BD Cytofix Buffer Cat. No. 554655; 37C for 10 min and permeabilized in BD Phosflow Perm Buffer III Cat. No. 558050 on ice 30 min . Cells were then stained with BD Phosflow PE Mouse Anti-Btk pY223 /Itk pY180 Cat. No. 562753 . Histograms showing BTK pY223 expression were generated for gated events with the forward and side-light scatter characteristics of intact cells using a BD FACSCanto II Flow Cytometer System. Panel 1b: Western blot analysis of BTK pY223 expressed by Ramos cells. Lysates from 1 million unstimulated C and Anti-IgM stimulated T Ramos cells were blotted using Purified Btk pY223 /Itk pY180 antibody 0.125, 0.063, and 0.032 mug/ml for Lanes 1, 2, and 3, respectively , HRP Goat Anti-Mouse Ig Cat. No. 554002 and a chemiluminescent detection system. BTK pY223 was identified as band of 77 kDa. Mouse Cells Panel 2a: Flow cytometric analysis of BTK pY223 expressed by mouse A20 cells. A20 cells mouse B lymphoma cell line were either not stimulated dashed line histogram or were stimulated solid line histogram with Rabbit Anti-Mouse IgG H+L Jackson, Cat. No. 315-005-003 at 37 C for 2 minutes. The cells were fixed, permed, stained and analyzed by flow cytometry as described above. Panel 2b: Western blot analysis of BTK pY223 expressed by mouse A20 cells. Lysates from 1 million untimulated C and Anti-IgM stimulated T A20 cells were blotted as described above with Purified Mouse Anti-Btk pY223 /Itk pY180 antibody at 0.032 mug/ml. Lower MW band at 42 kDa represents actin. Show More Analyses of BTK pY223 / ITK pY180 expression by Human and Mouse Cells. Human Cells Panel 1a: Flow cytometric analysis of BTK pY223 expressed by human Ramos cells. Serum-starved Ramos cells Human Burkitts lymphoma cell line were either not stimulated dashed line histogram or stimulated solid line histogram with Goat F ab 2 Anti-Human IgM Southern Biotech, Cat. No. 2022-14 at 37C for 2 minutes. Cells were fixed in BD Cytofix Buffer Cat. No. 554655; 37C for 10 min and permeabilized in BD Phosflow Perm Buffer III Cat. No. 558050 on ice 30 min . Cells were then stained with BD Phosflow PE Mouse Anti-Btk pY223 /Itk pY180 Cat. No. 562753 . Histograms showing BTK pY223 expression were generated for gated events with the forward and side-light scatter characteristics of intact cells using a BD FACSCanto II Flow Cytometer System. Panel 1b: Western blot analysis of BTK pY223 expressed by Ramos cells. Lysates from 1 million unstimulated C and Anti-IgM stimulated T Ramos cells were blotted using Purified Btk pY223 /Itk pY180 antibody 0.125, 0.063, and 0.032 mug/ml for Lanes 1, 2, and 3, respectively , HRP Goat Anti-Mouse Ig Cat. No. 554002 and a chemiluminescent detection system. BTK pY223 was identified as band of 77 kDa. Mouse Cells Panel 2a: Flow cytometric analysis of BTK pY223 expressed by mouse A20 cells. A20 cells mouse B lymphoma cell line were either not stimulated dashed line histogram or were stimulated solid line histogram with Rabbit Anti-Mouse IgG H+L Jackson, Cat. No. 315-005-003 at 37 C for 2 minutes. The cells were fixed, permed, stained and analyzed by flow cytometry as described above. Panel 2b: Western blot analysis of BTK pY223 expressed by mouse A20 cells. Lysates from 1 million untimulated C and Anti-IgM stimulated T A20 cells were blotted as described above with Purified Mouse Anti-Btk pY223 /Itk pY180 antibody at 0.032 mug/ml. Lower MW band at 42 kDa represents actin.