Metabolic labeling of cell proteins with [35S]-met 

(Protocol from An Zhou at Johns Hopkins, 1995)

1. Check that label is available - [35S]Met/Cys comes every other Friday (2 x 7.15 mCi total; 2 x 5 mCi Met) 

2. Sign up for labeling area - check with other people who may be labeling to avoid too many people doing complicated labelings at same time. 

3. Calculate how much label you need to dry down: 


[35S]Met is usually 1000 Ci/mmole 

[Met] in CSFM (Complete cell free medium) is 110 uM; for 15 to 30 min pulse, can go to 1 uM; for longer label need 5 uM 

LOG-OUT LABEL you are going to use from RADIATION LIVE notebook - for 

every 100 ul of [35S]Met used, log-out 1.4 mCi 

Dissolve [35S]Met in Met-/Cys- CSFM - store in bottom of GREY REVCO (next to autoclave) if not using immediately 

It takes 0.3 ml of medium to cover one well of a 4-well plate; use 0.4 ml if incubation time is longer than 15 to 30 min 

Can also do incubations in single well of 96 well plate - 15 min pulse of confluent AtT-20 cells gives 5 to 10 x 10^6 cpm cf TCA-precipitable material 

CHECK PIPETTES BEFORE AND AFTER YOU HANDLE LABEL - YOU ARE RESPONSIBLE FOR CLEANING UP ANYTHING FOUND DIRTY AFTER YOUR EXPERIMENT; USE FILTER TIPS. 

MANIPULATE LABEL AND LABELED MEDIUM IN HOT AREA IN 912, NEVER IN STERILE HOODS. 

4. In the labeling incubator, pre-equilibrate the Met-/Cys- CSFM, the [35S]MetlCys and the CSFM you will need; all incubations are done in lower chamber of designated incubator; all samples with [35S]Met are incubated inside another large square dish with a charcoal fiiter. 

When warming up the {35S]Met/Cys tube, keep it in another container - never place it directly into the water bath, since it could crack and leak, leaving a large mess you would not enjoy cleaning up. 

5. Incubate cells in Met-/Cys- CSFM for 5 to 10 min to deplete cellular stores of Met; longer is not better and can damage the cells. 

6. Discard Met-/Cys- CSFM and place [35S]Met/Cys/CSFM (~300 uCi/12 mm cell dish) onto cells. 300 uCi is also fine. May use 2 uM (that is, 600 uCi/300 uL, given the specific activity of 1000 Ci/mmol), when you need labeling really hot or have few cells. 

7. At end of labeling period, remove [35S]Met/Cys/CSFM - spin to remove cells if medium is to be re-used. Rinse cells with CSFM and either harvest or chase in a second aliquot of CSFM. 
  

8. Harvesting cell extracts: 

TES (10 mM)/mannitol/TX-100 (1%), pH 7.4: remove all medium; place 0.3 to 0.5 ml ice cold TMT onto plate; add PMSF and protease inhibitors (black X; 100X); scrape cells from plate with cut off yellow tip; transfer to microfuge tube and freeze on dry ice. 

Do 3 cycles of freezing and thawing to extract membrane proteins. 
Spin 3 to 5 min at top speed (14,000 rpm) in hot microfuge and transfer 
supernatant to new microfuge tube. 
Take 2 x 2 ul aliquots for TCA; freeze sample until you are ready to set up IPTs. 

Boiling SDS (best method): remove all medium; place 0.3 to 0.5 ml 1% SDS, 50 mM phosphate buffer, 20 mM 2-mercaptoethanol. 2 mM EDTA, pH 7.4 pre-heated to 100 ēC onto plate; transfer to microluge and boil 5 min. Spin 5 min and take 2 x 2 µl aliquots for TCA; freeze sample until you are ready to set up IPTs. Boil again to thaw - do not allow to thaw slowly. These samples need to be diluted with one-half volume 15% NP-40 before immunoprecipitation (the NP-40 binds up the SDS so it doesn't denature the antibody). 

5N Acetic Acid: for POMC and NPY. Wells are extracted into 0.2 ml 5 N acetic acid/2 mg/ml BSA plus 2 µl PMSF; extract is frozen and thawed three times and centrifuged for 5 min at top speed in hot microfuge. Supernatant is transferred to new microfuge tube and 2 x 2 µl taken for TCA precipitation. 

Lyophilize cell extract over night. 
Resuspend lyophilizate in 50 µl 1% acetic acid, add 1 µl PMSF and incubate on ice for 5 min 
Spin 5 min at top speed and transfer supernatant to fresh tube (for POMC need about 2 x 100 cpm/tube for IPT). 
Freeze on dry ice and lyophilize a second time. 
Resuspend in 280 µl Super E; add 3 µl PMSF, 3 µl inhibitor mix, 1 µl phenol red (to check pH) - if samples are yellow, pH is too low; add 1 µl Tris, pH 8.0 until color turns blue. Then ready to add antibody. 

9. Harvesting medium: remove medium from cells; spin 10 sec to remove debris; transfer supernatant to new tube; add PMSF and protease inhibitors; freeze until ready to set up IPT. No reason to do TCAs. 

10. TCA precipitation of cell extracts: to 5 µl BSA (40 mg/ml) in 1.5 ml microfuge tube, add 2 µl of sample and 0.7 ml 0.1 N NaOH. Wait 2 min and add 0.7 ml ice cold 25% TCA; let samples precipitate on ice for at least 15 min. Centrifuge 2 min and discard supernatant into appropriate liquid hot waste bottle. Add 0.5 ml TCA solubilizer solution and heat 5 min at 100 oC. Check that pellet has dissolved, allow to cool and then transfer to scintillation vial and get 1 min counts. 

11. Calculations: check how much of your label was incorporated into protein: take TCA cpm/ul x ul of sample; 1 mCi [35S] is approximately 2x109 cpm. You do not want to have more than 20% incorporation. Typically, one well (96 well plate) of AtT-20 cells will incorporate about 107 cpm of 35S-Met