Metabolic labeling experiments of recombinant proteins

Summary

Since host protein synthesis is essentially terminated when recombinant proteins are expressed, in vivo metabolic labeling is a sensitive method for detecting recombinant proteins.

Operation method

basic program

Materials and Instruments

Protein
Fetal Bovine Serum Methionine Cysteine
Culture flasks Centrifuges Rotors

Move

1. Inoculate 2.5 x ¹⁰⁶ cells in 60 mm muscle tissue culture dishes containing 4 ml of complete culture medium with 10% fetal bovine serum. Prepare a separate dish for infection with each putative recombinant virus and a control dish for infection with wild-type baculovirus. The cells were incubated at 27°C for 2 h. Cells were adhered to the wall. Aspirate the culture medium and add 1 ml of serum-free complete culture medium containing the recombinant virus or 1 ml of wild-type virus with a multiplicity of infections (MOI) of 5 to 10.
2. After removing the culture medium from each dish, add 3 ml of complete culture medium containing 10% fetal bovine serum to it and incubate at 27℃ for about 40 hours.3. Carefully remove the culture medium, wash the cells once with methionine-free or methionine- and cysteine-free culture medium, and add 1 ml of the same culture medium to which ^EXPRE35S35S has been added to 0.25-0.5 mCi/ml. The cells were incubated at 27℃ for 3~4 h. The cells were then incubated for 3~4 h.4. Transfer the cell and culture supernatants to a 15 ml polypropylene centrifuge tube and centrifuge at 1,000 g for 10 min at 4°C.
5. Perform SDS-PAGE to separate the proteins in the lysates. Add 20 to 40 μg of total cell protein or all of the immunoprecipitate per lane.6. Blot the proteins by autoradiography. Look at the autoradiograph to see if proteins of the expected molecular weight appear in cells infected with the recombinant virus (as opposed to the wild-type baculovirus).

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