The recombinant (Bβ1–66)2 fragment was produced in E. coli and purified as previously described9 (link). This fragment was treated with thrombin to produce fibrin-related (β15–66)2 fragment using the previously described procedure21 (link). Human recombinant receptor-associated protein (RAP) was expressed in E. coli and purified as previously described22 (link). Mouse anti-hVLDLR monoclonal antibodies 1H5, 5F3, and 1H1023 (link) were kindly provided by Dr. D. Strickland (University of Maryland Baltimore). Anti-His(C-term) antibody (anti-His tag mAb) conjugated with HRP was from Invitrogen (Carlsbad, CA). Goat secondary anti-mouse polyclonal antibodies conjugated with HRP and HRP substrate SureBlue TMB were from KPL (Gaithersburg, MD). Cyanogen bromide-activated Sepharose 4B and Thrombin CleanCleave kit were obtained from Sigma (St. Louis, MO).
Protocol detail
Preparation of Fibrin-Related Fragments and Antibodies
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Anti-His(C-term) antibody (anti-His tag mAb)
Cyanogen bromide-activated Sepharose 4B
Thrombin CleanCleave kit
Anti antibodies
Antibody
Cyanogen bromide
E coli
Fibrin
Goat
Human protein
Mouse
Sepharose 4b
Thrombin
Corresponding Organization :
Other organizations : University of Maryland, Baltimore
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Variable analysis
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- Treatment of (Bβ1–66)2 fragment with thrombin to produce fibrin-related (β15–66)2 fragment
- Production and purification of (Bβ1–66)2 fragment in E. coli
- Expression and purification of human recombinant receptor-associated protein (RAP) in E. coli
- Previously described procedures for production and purification of (Bβ1–66)2 fragment and human recombinant RAP
- Mouse anti-hVLDLR monoclonal antibodies 1H5, 5F3, and 1H10 provided by Dr. D. Strickland (University of Maryland Baltimore)
- Anti-His(C-term) antibody (anti-His tag mAb) conjugated with HRP from Invitrogen
- Goat secondary anti-mouse polyclonal antibodies conjugated with HRP and HRP substrate SureBlue TMB from KPL
- Cyanogen bromide-activated Sepharose 4B and Thrombin CleanCleave kit from Sigma
- None specified
- None specified
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