Instead of recovering RMs by ultracentrifugation, PPL-Ii translations (see Fig. 6 B) were subjected to an anti-Ii immunoprecipitation. Following translation, nine volumes of Triton IP buffer with anti-Ii antiserum (1:200) were added. Samples were incubated for 15 h at 4°C with constant agitation. Protein-A-Sepharose beads (Genscript) were added to 10% (v/v), and samples were incubated at 4°C for a further 2 h. Protein-A–Sepharose beads were then recovered by spinning at 13,000 g for 1 min and washed with Triton IP buffer before being heated at 70°C for 10 min in SDS sample buffer.
Protein a sepharose beads
Manufactured by GenScript
Sourced in United States
Protein-A–Sepharose beads are a type of affinity chromatography resin used for the purification of immunoglobulins (Igs) and other proteins that bind to Protein A. Protein A is a bacterial cell wall protein that has a high affinity for the Fc region of Igs, allowing for selective capture and isolation of Igs from complex mixtures.
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4 protocols using protein a sepharose beads
1
Anti-Ii Immunoprecipitation of PPL-Ii Translations
2
Immunoprecipitation of Protein Complexes
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Following carbonate extraction and recovery of the membrane fraction, pellets were resuspended in 20 µl of 1% (w/v) SDS and incubated for 10 min at 70°C. Ten volumes of Triton immunoprecipitation (IP) buffer [10 mM Tris-HCl pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5 mM PMSF, 1 mM methionine] with the appropriate antiserum (1:200) were added. Samples were incubated for 15 h at 4°C with constant agitation. Protein-A–Sepharose beads (Genscript) were added to 10% (v/v), and samples were incubated at 4°C for a further 2 h. Protein-A–Sepharose beads were then recovered by spinning at 13,000 g for 1 min and washed with Triton IP buffer before being heated at 70°C for 10 min in SDS sample buffer.
3
Characterizing CLEC14a-HSP70-1A Interaction
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The DNA sequences encoding CLEC14a-CTLD and fragments F1–F5 were amplified using polymerase chain reaction (PCR). The PCR products were subcloned to the two asymmetric Sfi I sites of a modified pCEP4 mammalian expression vector (Invitrogen) that contains the hinge and CH2-CH3 domains of human IgG1 at the 3′ end of the cloning site. Following producing each fragment in HEK293F cells, the fusion proteins were purified from the culture media using affinity chromatography with protein A Sepharose beads (GenScript). To determine the region through which HSP70-1A interacts with CLEC14a-CTLD, 0.1 µg of Fc fusion protein (CLEC14a-CTLD or F1–F5) were loaded onto a polyacrylamide gel for immunoblotting.
4
Recombinant VCAM-1-Fc Fusion Protein
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The DNA sequences encoding human VCAM-1-D6 (amino acids 511–595) and mouse VCAM-1-D6 (amino acids 511–595) were amplified by polymerase chain reaction (PCR). The PCR products were subcloned using two asymmetric SfiI sites into a modified pCEP4 mammalian expression vector (Invitrogen, Carlsbad, CA, USA) that contains the hinge and CH2-CH3 domains of human IgG1 at the 3′ end of the cloning site. The constructs were verified by DNA sequencing. The Fc fusion protein constructs were transfected into Expi293F™ cells using the ExpiFectamine™ Transfection Kit (Gibco). After seven days, the culture supernatants were collected, and the fusion proteins were purified using affinity chromatography with Protein A-Sepharose beads (GenScript, Piscataway, NJ, USA). After quantifying the protein concentrations using a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and performing dialysis in phosphate-buffered saline (PBS), the sample purity was analyzed by SDS-PAGE and Coomassie brilliant blue staining. The final pooled fractions were aliquoted and stored at −80 °C.
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