Anti-SHP1 antibody [EPR5519]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SHP1 antibody [EPR5519] (AB124942)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-SHP1 antibody [EPR5519] (AB124942)

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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SHP1 antibody [EPR5519] (AB124942)

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.

• WB

Lab

Western blot - Anti-SHP1 antibody [EPR5519] (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] (ab124942) 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='/en-us/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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• WB

Lab

Western blot - Anti-SHP1 antibody [EPR5519] (AB124942)

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] (ab124942) 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

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Western blot - Anti-SHP1 antibody [EPR5519] (AB124942)

All lanes:

Western blot - Anti-SHP1 antibody [EPR5519] (ab124942) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

Raji cell lysate at 10 µg

Lane 3:

lymph node lysate at 10 µg

Secondary

All lanes:

standard HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 68 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-SHP1 antibody [EPR5519] (AB124942)

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.

• WB

CiteAb

Western blot - Anti-SHP1 antibody [EPR5519] (AB124942)

SHP1 western blot using anti-SHP1 antibody [EPR5519] ab124942. Publication image and figure legend from Saidova, A., Potashnikova, D. M., et al., 2017, Cancer Med, PubMed 29125235.

ab124942 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab124942 please see the product overview.

Protein expression level of BCR components in B‐CLL ZAP70‐high and ZAP70‐low representative cases. (A) Immunoblot of signaling tyrosine kinases in ZAP70‐low sample (sample 1) and ZAP70‐high sample (sample 2), histone H3 was taken as a technical control. (B) Sample 1 (see A) analyzed by flow cytometry. Median fluorescence intensities of Zap70 in B‐CLL and T‐cell populations differ ×2.85‐fold; MFI(Zap70)T‐cells/MFI(Zap70)B‐CLL = 2.85. (C) Sample 2 (see A) analyzed by flow cytometry. Median fluorescence intensities of Zap70 in B‐CLL and T‐cell populations do not differ. MFI(Zap70)T‐cells/MFI(Zap70)B‐CLL = 1.

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