HRP Anti-SHP1 antibody [Y476]

3 product images

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  Western blot - HRP Anti-SHP1 antibody [Y476] (ab205427)

  False colour image of Western blot: HRP Anti-SHP1 antibody [Y476] staining at 1/5000 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab205427 was shown to bind specifically to SHP1. A band was observed at 70 kDa in wild-type THP-1 cell lysates with no signal observed at this size in PTPN6 knockout cell line Human PTPN6 knockout THP-1 cell line ab273717 (knockout cell lysate Human PTPN6 knockout THP-1 cell lysate ab273771). To generate this image, wild-type and PTPN6 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 before development with Optiblot (ECL reagent Anti-PKC delta (phospho S299) antibody [EPNCI119] ab133456) and imaged with 2 minutes exposure time. Secondary antibodies used were HRP conjugated Goat anti-Rabbit (H+L) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - HRP Anti-SHP1 antibody [Y476] (ab205427) at 1/5000 dilution

  Lane 1: Wild-type THP-1 cell lysate at 20 µg

  Lane 2: PTPN6 knockout THP-1 cell lysate at 20 µg

  Lane 3: Jurkat cell lysate at 20 µg

  Lane 4: K562 cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 68 kDa

  Observed band size: 70 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - HRP Anti-SHP1 antibody [Y476] (ab205427)

  IHC image of SHP1 staining in a section of formalin-fixed paraffin-embedded normal human tonsil tissue*, performed on a Leica BOND^TM. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab205427, 1/166 dilution, for 15 mins at room temperature. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay without primary antibody.
  

  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

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  Western blot - HRP Anti-SHP1 antibody [Y476] (ab205427)

  This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 3% milk before being incubated with ab205427 overnight at 4°C. Antibody binding was visualised using ECL development solution ECL Substrate Kit (High Sensitivity) ab133406.

  All lanes: Western blot - HRP Anti-SHP1 antibody [Y476] (ab205427) at 1/5000 dilution

  All lanes: Jurkat (Human) Whole Cell Lysate at 10 µg

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 68 kDa

  Observed band size: 65 kDa

  Exposure time: 90s