To detect protein antigens on apoptotic cells, cell membranes were labeled with D-Biotinyl-E-amidocaproic acid N-hydroxysuccinimide ester (Pierce Chemical Co.) according to the manufacturer's instructions. Western blot analysis was performed as described previously (12 (link)) and developed using an ECL kit (Amersham Biosciences) according to the manufacturer's instructions. The cells were incubated with 20% NHS or HGS, washed, and immunoprecipitated with rabbit IgG anti–human IgM adsorbed to protein G beads. Immunoprecipitated proteins were resolved by SDS-PAGE and detected by immunoblotting with streptavidin as described previously (12 (link)). Detection of ribosomal protein P0 (14 (link)) was used as a loading control.
Protocol detail
Detecting Apoptotic Cell Antigens via Western Blot
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Acid
Anti igg
Antigens
Apoptotic
Biotinyl n hydroxysuccinimide ester
Cell membranes
Cells
Human
Protein g
Proteins
Rabbit
Ribosomal protein p0
Sds page
Streptavidin
Western blot analysis
Corresponding Organization :
Other organizations : Cornell University, Hospital for Special Surgery, University of Washington
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Variable analysis
independent variables
- Cell membrane labeling with D-Biotinyl-E-amidocaproic acid N-hydroxysuccinimide ester
- Detection of protein antigens on apoptotic cells
- Western blot analysis of immunoprecipitated proteins
- Incubation of cells with 20% NHS or HGS
- Immunoprecipitation with rabbit IgG anti–human IgM adsorbed to protein G beads
- Detection of ribosomal protein P0 as a loading control
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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