Original
Quantitation of proteins expressed on the plasma membrane using cold-adapted proteases
Faraz Ahmad1, Sarah Kate Coleman2, Kai Kaila11,3, and Peter Blaesse1
1Laboratory of Neurobiology, University of Helsinki, Helsinki, Finland, 2Division of Biochemistry and Biotechnology, University of Helsinki, Helsinki, Finland, and 3Neuroscience Center, University of Helsinki, Helsinki, Finland
DOI: 10.2144/000113784

Abstract

Legend

⇒ATTENTION

*HINT

✋REST

Procedure

Cleavage of surface proteins of cultured cells

1. Wash the cells expressing your protein of interest with phosphate buffered saline (PBS) on ice.

2. Incubate the cells with the optimized concentration of cod trypsin in (PBS) for the required amount of time on ice (e.g., 1 mL PBS with cod trypsin for a 35 mm cell culture dish)

⇒ATTENTION: We recommend determining the optimal concentration and incubation time experimentally. A 1:50 dilution of the enzyme stock and a 60 min incubation can serve as a starting point. A 1:50 dilution corresponds to a final concentration of 2 U/mL (NOTE: We erroneously reported a concentration of 4 µU/mL in the paper due to a misconception of the concentration of the original cod trypsin stock solution).

3. Add trypsin inhibitor (phenylmethylsulfonyl fluoride, PMSF) up to 0.1 mM to stop the reaction (on ice, 5 min).

4. Harvest the cells in 1.5 mL tubes.

5. Pellet down cells and wash once with ice-cold PBS.

✋REST: To stop here, freeze the cell pellet and store at −20°C.

6. Homogenize the cells in a buffer appropriate for solubilizing membrane proteins (e.g., RIPA [100 mM NaCl, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 20 mM Tris-Cl, pH 8.0]).

*HINT: Use ∼10 µL of homogenization buffer/mg wet weight of cell pellet.

Cleavage of surface proteins in animal tissues

1. Prepare tissue slices of appropriate thickness (e.g., 400 µm for rat/mouse brain tissue).

2. Wash the slices with physiological solution (bubbled with oxygen or carbogen if required).

3. Incubate the slices with the optimized concentration of cod trypsin in the physiological solution for the required amount of time on ice (see step 2 above).

4. Add trypsin inhibitor (PMSF) up to 0.1 mM to stop the reaction (on ice, 5 min).

5. Transfer the slices into 1.5 mL tubes.

✋REST: To stop here, freeze the slices and store at −20°C.

6. Homogenize slices in a buffer appropriate for solubilizing membrane proteins.

*HINT: Use ∼10 µL of homogenization buffer/mg wet weight of tissue.

Calculation of the percentage of protein of interest expressed at the surface

7. Load ca. 30 µg of protein in the homogenate on SDS-polyacrylamide gels and blot it onto a PVDF/nitrocellulose membrane.

*HINT: The volume of homogenate loaded is related to the information given above regarding the volume of homogenization buffer to be used which determines the concentration of the proteins in the homogenate.

8. Probe with the desired antibody and quantitate the immunoreactive signal for the full-length and the cleaved fragment of your protein of interest.

9. The percentage of your protein of interest present in the membrane can be calculated from the ratio of the immunoreactive signal of the cleaved fragment and the full-length protein.

⇒ATTENTION: For accurate quantitation of the percentage, it is important that the same primary and secondary antibodies are used for detection of both the full-length protein and the cleavage fragment. In the case where your protein of interest has a high membrane expression compared with the intracellular pool, and the decrease in the full-length protein on cleavage with trypsin is reliably detectable, the decrease in the immunoreactive signal of the full-length protein in trypsin-treated compared with control samples can serve as an indirect measure of the membrane expression of the protein. The decrease in the full-length signal is the only measure for the surface expression of the protein of interest when the antibody does not detect a cleavage product (see also below).

Equipment needed

Glassware (Schott, Mainz, Germany)

Plastic ware (Sarstedt, Nümbrecht, Germany)

8453 UV-visible spectrophotometer (Agilent Technologies, Palo Alto, CA, U.S.)

Apparatus for performing SDS-PAGE (Bio-Rad, Hercules, CA, USA)

Electrophoretic transfer cell for blotting (Bio-Rad, Hercules, CA, USA)

Image reader for detection of immunoreactive signal (LAS 3000, Fujifilm, Tokyo, Japan)

Troubleshooting

Problem: There seems to be no cleavage of the protein of interest even when it is expressed on the membrane.

Possible cause and solution: The enzyme concentration is too low. Optimize the enzyme concentration.

Possible cause and solution: The cleavage site is not accessible to the enzyme (e.g., intracellular cleavage sites, conformational constraints, or glycosylation) or there is no cleavage site. Use bioinformatics algorithms (such as HMMTOP) to make predictions about the presence and position of cleavage sites. Try using another cold-adapted protease if it is likely that there is no (accessible) trypsin cleavage site.

Possible cause and solution: The enzyme has lost its activity (e.g., due to freeze-thaw cycles). Incubate the protein homogenate with cod trypsin and check for cleavage of proteins.

Problem: There is digestion of intracellular proteins like tubulin.

Possible cause and solution: The enzyme concentration is too high. Lower the concentration of the enzyme to preserve the integrity of the cells.

Possible cause and solution: The exposure time of trypsin to the cells is too high. The cleavage time should be optimized and reduced appropriately.

Problem: There is cleavage of your protein of interest (which is observable as a decrease in the immunoreactive signal of the full-length protein) but a cleaved fragment is not detectable.

Possible cause and solution: The antibody for your protein of interest is against an extracellular epitope and the cleaved fragment is present in the medium after cleavage of your protein by cod trypsin. Concentrate the extracellular medium as required and analyze the presence of the cleaved fragment.

Possible cause and solution: The epitope is not intact after cod trypsin cleavage. Use a different antibody if available.

Possible cause and solution: The cleaved fragment which is detectable by your antibody is intracellular but is rapidly degraded. If available, use an antibody that recognizes another epitope. It might detect a more stable fragment of the protein.

Reagents

Cod trypsin/Penzyme (Zymetech, Reykjavik, Iceland)

Protease inhibitor cocktail (Roche, Mannheim, Germany)

Phenylmethylsulfonyl fluoride (PMSF; Sigma, Steinheim, Germany)

DC protein assay kit (Bio-Rad, Hercules, CA, USA)

ECL Western blotting substrate (Pierce, Rockford, IL, USA)

All other chemicals were of analytical grade and were obtained from other local suppliers.

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