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Review on EMD Millipore #05-321 Monoclonal Mouse anti-Phosphotyrosine antibody

  • WB Rating: 4
Dec 30, 2011 by
  • Application: Western blot
  • Non-traditional application details: SNAP i.d.
  • Application rating: 4 - Excellent (Publishable / Ideal reagent)
  • Specificity: Specific
  • Sensitivity: Sensitive
  • Sample: EGFR-Transfected, EGF-stimulated CHO-S Cells
  • Buffer: PBST
  • Dilution: 1/1000

Other antibodies:

  • Santa Cruz sc-2062 (Dilution: 1/3000)

Other experiment details:

CHO-S cells were transfected with C-terminally HA-tagged EGFR, and stable colonies were selected. Cells were stimulated with 100 ng/mL EGF for 5 min. in 24-well plates, then lysed with RIPA buffer. Unstimulated control wells were also lysed in the same way. Anti-HA antibody was added to the lysates and incubated overnight with Protein G beads. The beads were then washed, eluted with SDS-PAGE loading buffer, and precipitated protein run on SDS-PAGE gels for Western blotting.

The arrow is at the expected position of EGFR. The doublet between 20-30 kDa is due to Protein G, which is also eluted from the resin and retains some antibody binding ability. The protein G band binds primary/secondary Western antibodies and is therefore detected.

Note that the given antibody dilutions are appropriate only for Westerns using the SNAP i.d. device (Millipore). For a standard western, multiply the dilution by 3/10.

Additional documents:

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About EMD Millipore

EMD Millipore Bioscience delivers new technologies & workflow solutions that enable researchers to understand complex biological systems & identify new targets.