DPP4 staining ( em upper panel /em ?) and MERS-CoV-S1 binding ( em lower panel /em ?) are shown Virus Infection Assay Transfect pcDNA-DPP4 plasmid or plasmid containing the gene under investigation in Cos-7 and empty plasmid as control. After 24 h of transfection, wash the cells with Cos-7 growth medium and incubate the cells with virus under investigation, Tirabrutinib e.g., MERS-CoV-EMC for 1 h. Wash the cells two Tirabrutinib times with Cos-7 growth medium containing 1 % FCS to remove any unbound virus and after final wash add 3 ml of fresh medium. Incubate the cells at 37 C with 5 % CO2 for 24 h. Fix the cells with 4 % formaldehyde solution for 10 min. Wash the cells three times with PBS. Add 500 l of 70 %70 % ethanol and keep the plate at 4 C until immunofluorescent staining. Wash the cells three times with PBS. Add 200 l of 10 %10 % normal goat serum or serum corresponding to the species from which the secondary antibody in step 14 is derived. Incubate the cells at 37 C for 30 min. Remove the 10 %10 % normal goat serum and add 200 l of any antibody to a specific virus Tirabrutinib protein, for example rabbit-anti-SARS-CoV nsp4 (5 g/ml) is cross-reactive for MERS-CoV-EMC. Incubate the cells at 37 C for 1 h. Wash the cells three times with PBS. Add 200 l of goat anti-rabbit serum conjugated with FITC (5 g/ml). Incubate the cells at 37 C for 1 Itgb1 h. Wash the cells three times with PBS and analyze using a fluorescent microscope (Fig. to identify and characterize the receptor for the Middle East respiratory syndrome coronavirus. This technique can be adapted to identify other viral receptors. cells by adding 2C5 l of ligation mixture to 50 l competent cells. Incubate on ice for 30 min. Heat-shock cells for 30 s at 42 C in a thermocycler or water bath. Add 250 l of SOC medium. Incubate at 37 C for 1 h while shaking. Plate 100 l on prewarmed LB Amp plates. Incubate at 37 C overnight. Next day, pick colonies using a sterile toothpick for colony PCR screening and storage. Colony PCR Transfer a small amount of a colony into 25 l of LB medium. Make a PCR mix as follows: 2 l 10 PCR polymerase buffer, 1 l 10 mM dNTPs, 0.6 l 10 mM forward primer, 0.6 l 10 mM reverse primer, 1 l of the colony mix, 1 l PCR polymerase, 13.8 l dH2O. Incubate the PCR mix for 30 cycles at 94 C for 20 s, 58 C for 30 s, 72 C for 2 min, with a final extension at 72 C for 10 min. Analyze the PCR products by standard agarose gel electrophoresis. Inoculate PCR positive clones in 2 ml LB Amp medium and to grow at 37 C for ~8 h while shaking. From this 2 ml culture, inoculate 500 l into a 500 ml of LB Amp medium. Allow the bacteria to grow ~8 h at 37 C while shaking. Next day, extract plasmid DNA from the bacteria using a maxi prep DNA kit, according to manufacturers instructions. Perform a restriction digest and analyze the products by standard agarose gel electrophoresis to confirm the plasmid DNA is correct. Determine DNA concentration using a NanoDrop or spectrophotometer and prepare a DNA stock of 1 1 g/l. Large-Scale Expression and Purification of S1-Fc Fusion Proteins Seed HEK-293T cells in 20T175 flasks in 40 ml of 293T cell growth medium and incubate at 37 C with 5 % CO2 for approximately 24 h until 60C70 % confluent. Prepare a working stock of 1 1 mg/l PEI. This can be kept at 4 C. Two hours prior to transfection, remove medium from 293T cells and replace with 30 ml of fresh prewarmed 293T cell growth medium. For each T175 flask, prepare the DNA transfection solution as follows: add 18 l of 1 1 g/l pCAGGS-MERS-CoV-S1-Fc plasmid DNA (Subheading 3.5, step 10) to 3 ml of serum-free DMEM and mix by pipetting. Add 54 l of 1 1 mg/l PEI to the transfection solution and mix. Incubate at room temperature for 30 min. Add the DNA/PEI complex dropwise to the T175 flask and gently swirl to mix. Incubate cells 4C12 h (determine experimentally). Aspirate the medium from the transfected cells and replace with 40 ml of HEK-293T expression medium, incubate at 37 C with 5 % CO2 for 6 days. Prepare 50 % (w/v) protein-A sepharose beads: Add 0.25 g of protein-A sepharose CL-4B to a tube, add 10 ml PBS to form a slurry, centrifuge for 2 min at 2,000??and resuspend in 1.4 ml PBS per tube (50 % w/v), the final volume will be ~2.8 ml. Collect the expression medium from the transfected HEK-293T cells into 50 ml tubes and centrifuge at 2,850??for 10 min to remove cell debris. Transfer medium to new 50 ml tubes and centrifuge again at 2,850??for 15 min. Transfer cleared medium to new 50 ml tubes and keep it on ice; take a 100 l aliquot and store at ?20 C. Add 0.5 ml of washed protein-A sepharose beads (50 % w/v) Tirabrutinib and 800 l of 1 1 M TrisCHCl pH 8.0 to each 40 ml supernatant to neutralize the pH and incubate overnight, rotating at 4 C (for 15 min (see Note 6). Pool all the protein-A sepharose beads together in a 50 ml tube and wash three times with 10 ml PBS. After the final centrifugation, resuspend the protein-A sepharose beads in 1 ml of 0.5 M acetic acid pH 3 elution buffer and incubate for 1 min at room temperature. Centrifuge the protein-A sepharose beads at 14,000??for 10 min and transfer the supernatant to a 1.5 ml tube. Repeat steps 17 and 18 twice more to elute any remaining S1-Fc protein from the protein-A sepharose beads. To remove any remaining protein-A sepharose beads in the supernatant repeat step 18 once and transfer supernatant to a fresh tube. To neutralize the pH.