All lanes : Anti-SHP1 antibody [EPR5519] (ab124942) at 1/1000 dilution

Lane 1 : Wild-type THP-1 cell lysate
Lane 2 : PTPN6 knockout THP-1 cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : K562 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 68 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab124942 observed at 70 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab124942 was shown to react with SHP1 in wild-type THP-1 cells in Western blot with loss of signal observed in PTPN6 knockout sample. Wild-type THP-1 and PTPN6 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab124942 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

ab124942, at a 1/1000 dilution, staining SHP1 in paraffin-embedded Human tonsil tissue by immunohistochemistry

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunocytochemistry/Immunofluorescence analysis of Raji (human Burkitt's lymphoma) cells labelling SHP1 with purified ab124942 at a dilution of 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (ab150077) at dilution of 1/1000 was used as the secondary antibody. Nuclei counterstained with DAPI (blue).

Secondary Only Control: PBS was used instead of the primary antibody as the negative control.

All lanes : Anti-SHP1 antibody [EPR5519] (ab124942) at 1/1000 dilution

Lane 1 : Jurkat cell lysate
Lane 2 : Raji cell lysate
Lane 3 : lymph node lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : standard HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 68 kDa

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling SHP1 with purified ab124942 at 1/100 dilution (10 µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cells without incubation with primary antibody and secondary antibody (Blue).

Equilibrium disassociation constant (K_D)
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