Anti-SHP1 antibody [EPR5519] - BSA and Azide free

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHP1 antibody [EPR5519] - BSA and Azide free (AB240949)

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

This data was developed using the same antibody clone in a different buffer formulation containing tissue culture supernatant, PBS, BSA, glycerol, and sodium azide (ab124942)

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SHP1 antibody [EPR5519] - BSA and Azide free (AB240949)

Immunocytochemistry/Immunofluorescence analysis of Raji (human Burkitt's lymphoma) cells labeling SHP1 with purified ab124942 at a dilution of 1/500. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-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.

This data was developed using the same antibody clone in a different buffer formulation containing tissue culture supernatant, PBS, BSA, glycerol, and sodium azide (ab124942)

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-SHP1 antibody [EPR5519] - BSA and Azide free (AB240949)

This data was developed using ab124942, the same antibody clone in a different buffer formulation.Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling SHP1 with purified ab124942 at 1/100 dilution (10μg/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).

• WB

Lab

Western blot - Anti-SHP1 antibody [EPR5519] - BSA and Azide free (AB240949)

This data was developed using ab124942, the same antibody clone in a different buffer formulation.

Western blot : Anti-SHP1 antibody [EPR5519] ab124942 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 68 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in PTPN6 CRISPR-Cas9 Edited MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween 20 (TBS-T) before incubation with primary antibodies overnight at 4 C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-SHP1 antibody [EPR5519] (<a href='/ko/products/primary-antibodies/shp1-antibody-epr5519-ab124942'>ab124942</a>) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human PTPN6 knockout MCF7 cell line (ab287732) at 20 µg

Lane 3:

THP-1 at 20 µg

Lane 4:

SH-SY5Y at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 68 kDa

Observed band size: 68 kDa

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• WB

Lab

Western blot - Anti-SHP1 antibody [EPR5519] - BSA and Azide free (AB240949)

This data was developed using the same antibody clone in a different buffer formulation (ab124942).

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.

All lanes:

Western blot - Anti-SHP1 antibody [EPR5519] (<a href='/ko/products/primary-antibodies/shp1-antibody-epr5519-ab124942'>ab124942</a>) at 1/1000 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

PTPN6 knockout THP-1 cell lysate at 20 µg

Lane 2:

Western blot - Human PTPN6 knockout THP-1 cell line (<a href='/ko/products/cell-lines/human-ptpn6-knockout-thp-1-cell-line-ab273717'>ab273717</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

K562 cell lysate at 20 µg

Predicted band size: 68 kDa

Observed band size: 70 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-SHP1 antibody [EPR5519] - BSA and Azide free (AB240949)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.