Overview

  • Product name

    Cell Surface Protein Isolation Kit
  • Sample type

    Adherent cells, Suspension cells
  • Assay time

    3h 00m
  • Product overview

    Cell surface protein isolation kit (ab206998) enables the isolation of cell surface proteins for various downstream applications such as western blotting and other structural and functional studies. The cell surface protein isolation kit provides a simple and efficient method for the isolation of cell surface proteins.


    In this method, cells are first labeled with Sulfo-NHS-SS-Biotin, an amine-reactive, thiol-cleavable, biotinylation reagent. Cells are subsequently lysed and the labeled cell surface proteins are isolated using streptavidin beads. The bound proteins are then released from beads by incubating with DTT solution. The biotinylation reagent is cell membrane-impermeable, with an extended spacer arm to reduce steric hindrances associated with streptavidin binding.


    Cell surface protein isolation protocol summary:
    - remove culture media and wash cells
    - add biotin solution and incubate for 30 min
    - add quenching solution and incubate for 5 min
    - collect cells and spin at 500 g for 3 min
    - wash cell pellet twice
    - resuspend pellet in lysis buffer and incubate for 30 min
    - spin at 10,000 g for 2 min and retain supernatant
    - add supernatant to streptavidin beads and incubate for 1 hr
    - spin at 800 g for 1 min
    - wash beads twice and spin at 800 g
    - add elution buffer and incubate for 30 min
    - spin down beads and collect supernatant for analysis

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 10 tests
    DTT 1 x 100µl
    Lysis Buffer 1 x 6.5ml
    PBS Tablet 1 x 3 tablets
    Quenching Solution 1 x 10ml
    Streptavidin Beads 1 x 1.5ml
    Sulfo-NHS-SS-Biotin 5 vials
    TBS Tablet 1 tablet
    Wash Buffer 1 x 12ml

Images

  • western blot analysis of cell surface proteins isolated using ab206998  Cell surface proteins were isolated from HeLa cells in the presence (+) and absence (-) of Sulfo-NHS-SS-Biotin. The blots were probed with antibodies to Endoplasmic Growth Factor Receptor (EGFR), a plasma membrane marker (top) and Calnexin, a cytosolic marker (bottom).

  • western blot analysis of isolated cell surface proteins  HeLa cells were used to isolate the cell surface proteins, in the presence (+) and absence (-) of Sulfo-NHS-SS-Biotin. (b) Integrin β1 and Hsp90 as plasma membrane and cytosolic markers, respectively. Isolation was performed following the kit protocol.

Protocols

References

ab206998 has not yet been referenced specifically in any publications.

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