Metabolic labeling in vivo is a sensitive way to detect recombinant proteins, since at the time the recombinant protein is actually expressed, host protein synthesis is actually terminated.
Thus, many label is included into late-viral-specific proteins, such as protein of awareness.
The most very popularly used procedure for radiolabeling proteins will be the incorporation of [35S]methionine and also [35S]cysteine, both which are essential amino acids.
Intended for better results, the intracellular pool worth mentioning two amino acids must be depleted prior to help radiolabeling.
This is usually achieved by preincubating that cells in methionine/cysteine–free method for 30 minutes. The efficiency regarding incorporation depends on the amount of methionines and cysteines from the particular protein of great interest.
After labeling, the cells are lysed.
The proteins are resolved by SDS-PAGE in addition to visualized by autoradiography.
Supplemental Materials
| Wild-type baculovirus | Methionine-free or methionine-free/cysteine-free Grace’s insect cell culture medium (Invitrogen) |
| EXPRE35S35S, containing [35S]methionine and [35S]cysteine (>1000 Ci/mmol; Du Pont NEN) | Additional reagents and equipment for one-dimensional SDS-PAGE and autoradiography (APPENDIX 3A) |
1. Seed 2. 5 × 106 tissues into 60-mm tissue culture plates formulated with 3 ml TNM-FH method with 10% FBS. Prepare one plate to become infected with every putative recombinant virus your decide one control plate for being infected with wild-type baculovirus.
(Prepare one plate for every viral stock to be tested and one plate as a possible uninfected control.)
2. Incubate 1 hr at 27°C to allow for cells to fix.
Aspirate medium, adding 1 ml recombinant trojan or 1 ml carrier containing wild-type malware at an MOI regarding 5 to 10.Incubate 1 human resources at room heat.
3. Remove this medium from every plate by aspiration. Add 3 ml TNM-FH/10% FBS medium to the cells and incubate TWENTY FOUR to 48 hours at 27°C.
4. Carefully remove carrier from each eating plan, then rinse tissue once with methionine-free and also methionine-free/cysteine-free medium.
Increase 1 ml methione-free or even cysteine-free medium for you to each plate.
Incubate debris 30 min at 27°C, then bring 0. 25 for you to 0. 5 µCi EXPRE35S35S per registration and incubate Three or four hr at 27°C.
（It is important to hold the plates at an opinion and remove in addition to add medium in one corner so as not to dislodge the cells from your monolayer.）
5. Transfer cells as well as culture supernatant via each plate to your separate 15-ml polypropylene centrifuge television and centrifuge 15 min at 1000 × g (2000 rpm in a GH-3. 7 rotor), 4°C.
6. Process and review the cells (if that recombinant protein seriously isn't secreted) or that supernatant (if the recombinant protein can be secreted).
7.Create in your mind proteins by autoradiography (APPENDIX 3A). Look the autoradiogram regarding protein of the actual expected molecular pounds that appears within cells infected by using recombinant baculovirus however , not with wild-type baculovirus.
Related reading：
Steps For Overproducproteins Using The Baculovirus System
Introduction To Baculovirus Expression System