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Tpx ll-Xpress total protein

^ streptavidin extracted

HRP-SA (reduced)

Fig. 4. Detection of BioGEE incorporation into protein, and selectivity for reactive cysteines.

(A) HeLa cells were loaded with 0.5 mM BioGEE for 1 hr and then exposed to H202 (0,0.12,0.25,0.5, and 1 mM) for 15 min. A soluble protein extract was obtained as described in text and 40 /¿g of protein from each extract was resolved by SDS-PAGE under either nonreducing or reducing conditions. The proteins were transferred to nitrocellulose and the blots were stained with Ponceau S. The blots were then blocked with milk and probed with horseradish peroxidase-conjugated streptavidin (HRP-SA).

(B) HeLa cells were transfected with cDNAs encoding epitope-tagged wild-type or mutant annexin II and thioredoxin peroxidase II (Tpx II). The annexin II mutant contained a glycine substitution for the reactive cysteine at amino acid 9 (see Ref. 21), and the Tpx II mutants contained serine substitutions at the active site cysteines Cys-52 (reactive, see Ref. 26) and Cys-173 (not reactive). The cells were exposed to the indicated oxidative stress (TNF-a/cycloheximide 3-hr stimulation; H2O2, 15-min exposure), and a soluble protein extract was prepared as described in text. Biotin-containing proteins were affinity purified, blotted to nitrocellulose, and probed with antibodies against the indicated epitope tags.

(NP-40), 0.1% (w/v) sodium dodecyl sulfate (SDS), sodium deoxycholate (0.5 mg/ml), 150 mM NaCl, and 50 mM Tris-HCl (pH 7.5)], and hypotonic lysis buffer [1 mM EDTA, 1 mM EGTA, 50 mM Tris-HCl (pH 7.0)]. Standard equipment, solutions, and materials for SDS-polyacrylamide gel electrophoresis (PAGE) and transfer of proteins to nitrocellulose are also needed.

Method for Loading Cells with Biotinylated Glutathione Ethyl Ester

To load cells in culture we typically resolubilize BioGEE in culture medium at a concentration of approximately 0.5 mM, filter the medium, and then add it directly to cells. To estimate the concentration of BioGEE, one pellet from each synthesis lot should be dissolved in PBS and the concentration determined with Ellman's reagent as described above. In our experience, good results can be obtained by loading cells from 1 hr to overnight and in the absence or presence of up to 10% (v/v) fetal bovine serum (FBS). Although there is some basal incorporation of BioGEE into protein in unchallenged cells, the effect of oxidative stress on incorporation is obvious even after 16 to 18 hr of loading. Basal incorporation of BioGEE is probably a steady state phenomenon as oxidative stress appears to increase labeling of many of the same bands detected in the basal state. In addition, the extent of BioGEE incorporation in the basal state appears to be protein specific as we cannot detect basal incorporation of BioGEE into glyceraldehyde-3-phosphate dehydrogenase (GAPDH),21 a protein for which incorporation is readily detectable after oxidative stress.

Because BioGEE is likely to enter the cell by passive diffusion, we anticipate that it will be possible to load a wide variety of cell types and tissues with the reagent. The efficiency of loading might vary, however, depending on how BioGEE is metabolized by the individual cell type. For example, results of one study indicate that the diethyl ester of glutathione crosses the plasma membrane in both directions more rapidly than the monoethyl ester.25 In some human cell types the diethyl ester is rapidly metabolized to the monoester, resulting in a partial trapping and concentration of the reagent inside of the cell. However, the rate of conversion of the monoethyl ester to GSH is slow in these same cells. In our experience oxidant-induced incorporation of BioGEE declines rapidly once the reagent is washed out of the culture medium, probably because of efflux of incompletely deesterified label from the cell. Therefore we typically leave the reagent in the culture medium during stimulation. The need to do this might, however, vary from one cell type to the next, depending on how rapidly the cell deesterifies the second carboxylate.

26 S. Hirotsu, Y. Abe, K. Okada, N. Nagahara, H. Hon, T. Nishino, and T. Hakoshima, Proc. Natl. Acad. Sci. U.S.A. 96, 12333 (1999).

Method for Harvesting Proteins: For Both Blotting and Affinity Purification

1. Wash the cells twice with ice-cold PBS and once with ice-cold PBS containing 50 mM /V-ethylmaleimide (NEM) to scavenge any remaining BioGEE.

2. Harvest cells by scraping in RIPA or hypotonic lysis buffer containing NEM or iodoacetamide (IAM) at 50 mM concentration and protease inhibitors. We have obtained good results with both NEM and IAM. However, it is important to note that we do not know of a peptide-mapping resource that includes NEM-modified cysteines, and therefore IAM should be used if the goal of the experiment is to identify a protein by peptide mapping.

3. For cells harvested in RIPA buffer, homogenize by passing the lysate several times through a 22-gauge needle. For cells harvested in hypotonic buffer, lyse by freezing in a dry ice-ethanol bath and thawing at room temperature.

4. Centrifuge the lysates for 10 min at 12,000g and 4° to pellet insoluble material and transfer the supernatant to a fresh tube.

5. Determine the protein concentration in the supernatant, using BCA or other standard methods.

Method for Detection of Biotinylated Glutathione Ethyl Ester Incorporation into Protein

Standard laboratory Western blotting and immunoprecipitation protocols can be easily adapted for assaying BioGEE incorporation into total or selected proteins. It is, of course, important to keep in mind that BioGEE is incorporated via a disulfide bond and therefore reducing agents must be omitted from buffers and solutions unless the intention is to remove the label. We have obtained good results with both freshly prepared and frozen (—80°) lysates, and from as little as 5 //g of soluble protein. For detection of biotin in proteins blotted to nitrocellulose membranes, we typically block the membranes with 5% (w/v) nonfat dry milk in PBS-T for 1 hr at room temperature. The membranes are then probed with HRP-conjugated streptavidin at 1 fig/ml for 1 hr at room temperature. The blots are washed five times with PBS-T and developed with HRP chemiluminescent substrate according to the manufacturer protocol. It is important to be aware that biotin-containing proteins such as carboxyltransferases might also be detected by this protocol. However, because incorporation of biotin via a disulfide is unique to BioGEE, labeling with BioGEE can be confirmed by running a duplicate blot under reducing conditions (Fig. 4A).

BioGEE incorporation into individual proteins can be determined by probing blots of immunoprecipitates with streptavidin, or by probing blots of affinity-purified proteins (described below) with antibodies. In our experience the former approach is both more sensitive and more reliable than the latter.

Affinity Purification of Biotinylated Glutathione Ethyl Ester-Labeled Proteins

Perhaps the greatest strength of the method described here is that it provides a means to affinity purify proteins on the basis of their propensity to undergo redox modification at select amino acid residues. We have used the simple two-step scheme described below to successfully identify, by mass spectrometric peptide mapping and peptide sequencing, proteins purified from as little as 5 mg of soluble protein (Fig. 5). In the protocol described below proteins covalently bound to biotin are extracted in batch. The protocol can be readily adapted to a column if so desired.

Materials

Streptavidin-agarose can be obtained from Sigma and Centricon-10 ultrafiltration units can be obtained from Amicon (Beverly, MA). Biotin-blocked strepta-vidin is prepared by suspending 1 ml of streptavidin-agarose in 10 ml of (D-biotin 3 mg/ml in PBS), incubating the mixture for at least 1 hr at room temperature, and then washing the beads extensively with PBS. For in-gel protein detection we use the SilverXpress silver staining kit (Invitrogen, Carlsbad, CA) and GelCode Blue Coomassie staining reagent (Pierce).

Method

1. Obtain protein extracts as described above.

2. Add biotin-blocked streptavidin-agarose (50 ¿il/mg of soluble protein) to the extract and incubate for 30 min at 4° with gentle shaking.

3. Pellet the beads by brief centrifugation and transfer the supernatant to a fresh tube.

4. Add 100 /til of agarose-conjugated streptavidin per milligram of protein and incubate for 2 hr at 4° with gentle shaking.

5. Pellet the beads by brief centrifugation and remove the supernatant.

6. Wash the beads five times with approximately 10 volumes of ice-cold RIPA buffer.

7. Wash the beads two times with 10 volumes of room temperature PBS containing 0.1% (w/v) SDS.

8. Resuspend the agarose pellet in 1 volume of PBS-0.1% (w/v) SDS and incubate for 30 min at room temperature with gentle shaking.

9. Pellet the agarose and save the supernatant to be run in parallel as a —DTT control.

10. Resuspend the agarose pellet in 1 volume of PBS-0.1% (w/v) SDS containing 10 mM DTT and incubate for 30 min at room temperature with gentle shaking.

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