Detecting TAPBPR and MHC I Components

TAPBPR was detected using either PeTe4, a mouse monoclonal antibody (mAb) specific for the native conformation of TAPBPR, raised against amino acids 22–406 of human TAPBPR (Boyle et al., 2013 (link)) that does not cross-react with tapasin (Hermann et al., 2013 (link)), or ab57411, a mouse mAb raised against amino acids 23–122 of TAPBPR that is reactive to denatured TAPBPR (Abcam, UK). Tapasin was detected using Pasta-1 (Dick et al., 2002 (link)), or with R.gp48N, a rabbit polyclonal antibody specific for tapasin (Sadasivan et al., 1996 (link)) (kind gifts from Peter Cresswell, Yale University School of Medicine). MHC I heavy chains were detected using mAb HC10 (Stam et al., 1986 (link)) and mAb HCA2 (Stam et al., 1990 (link)). β₂m was detected using a rabbit polyclonal antibody (Dako, UK). Cell surface MHC I molecules were detected using W6/32, a pan-MHC I mAb that recognizes a conformational epitope on the α2 domain of MHC I, in a manner dependent on presence of β₂m and peptide (Barnstable et al., 1978 (link)). Calnexin was detected via western blot analysis using the rabbit polyclonal ADI-SPA-860 (Enzo Life Sciences, UK). A mouse IgG2a isotype control was also used as a control (Sigma-Aldrich).

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Ilca F.T., Drexhage L.Z., Brewin G., Peacock S, & Boyle L.H. (2019). Distinct Polymorphisms in HLA Class I Molecules Govern Their Susceptibility to Peptide Editing by TAPBPR. Cell Reports, 29(6), 1621-1632.e3.

Publication 2019

PeTe4 ab57411 ADI-SPA-860 mouse IgG2a isotype control

Amino acids Antibody Calnexin Cell Epitope Gifts Human Igg2a Isotype Mhc i molecules Monoclonal antibody Mouse Pasta Peptide Rabbit Tapasin Western blot analysis

Corresponding Organization : University of Cambridge

Other organizations : University of Freiburg, Cambridge University Hospitals NHS Foundation Trust

Top 5 similar protocols

Variable analysis

independent variables

Use of PeTe4 mouse monoclonal antibody (mAb) specific for the native conformation of TAPBPR
Use of ab57411 mouse mAb raised against amino acids 23-122 of TAPBPR that is reactive to denatured TAPBPR

dependent variables

Detection of TAPBPR
Detection of tapasin
Detection of MHC I heavy chains
Detection of β2m
Detection of cell surface MHC I molecules
Detection of calnexin

control variables

Amino acids 22-406 of human TAPBPR used to raise PeTe4 mAb
Amino acids 23-122 of TAPBPR used to raise ab57411 mAb
Use of Pasta-1 and R.gp48N antibodies to detect tapasin
Use of HC10 and HCA2 mAbs to detect MHC I heavy chains
Use of rabbit polyclonal antibody to detect β2m
Use of W6/32 pan-MHC I mAb to detect cell surface MHC I molecules
Use of rabbit polyclonal ADI-SPA-860 antibody to detect calnexin
Use of mouse IgG2a isotype control

positive controls

Use of Pasta-1 and R.gp48N antibodies to detect tapasin
Use of W6/32 pan-MHC I mAb to detect cell surface MHC I molecules

negative controls

Use of mouse IgG2a isotype control

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