Proteinase K protection assay

1. Seed cells (e.g. HEK293T cells) in a 10cm dish and culture cells to confluence (1×107cells).

2. Collect the cells by a scraper, followed by centrifugation at 600×g for 5 min at 4°C.

3. Wash the cells twice with ice-cold PBS, and suspend them in 250 µl (2.5 times the volume of cell pellet) of HB1 lysis buffer.

4. Homogenize the cells with ~20 strokes using a 1 mL syringe with 22-G needle. Stain 2μl cell lysate with trypan blue and visualize through a microscope. If less than 90% cells are lysed, add more strokes. (Note: Times of strokes varies among different types of cells.)  

5. Centrifuge the sample at 1000×g for 10 min at 4°C to recover the postnuclear supernatant (PNS).

6. Centrifuge the PNS at 25,000×g for 10min at 4℃ to collect the 25K membrane pellet.

7. Wash membrane pellet with 500μl B88 buffer and centrifuge at 25,000×g for 10min at 4℃.

8. Resuspend membrane pellet in 100ml of B88 and divide into three 30 ml of aliquots. Of the three aliquots, one is control, the other two are incubated with 10-30mg/mL proteinase K (1mg/ml stock) with or without 0.5% Triton X-100 (10% stock) on ice for 30 min.

9. Stop the reactions by adding 3μl 100mg/mL AEBSF/PMSF on ice for 10 min.

10. Add boiled 3×SDS loading buffer and boil the sample for 10 min followed by Western blot analysis.

HB1 lysis buffer

20mM HEPES-KOH, pH 7.2

1mM EDTA

400mM sucrose

Protease inhibitors

Phosphatase inhibitors

0.3 mM DTT

B88 buffer

20mM HEPES-KOH, pH 7.2

250mM sorbitol

150mM potassium acetate

5mM magnesium acetate

Reference

1.    Min Zhang, Lei Liu, Xubo Lin, Yang Wang, Qing Guo, Ying Li, Shulin Li, Yuxin Sun, Di Zhang, Xiachen Lv, Li Zheng and Liang Ge. (2020). A translocation pathway for vesicle-mediated unconventional protein secretion. Cell  181:637-652.

2. Zhang, M., Kenny, S.J., Ge, L., Xu, K., and Schekman, R. (2015). Translocation of interleukin-1β into a vesicle intermediate in autophagy-mediated secretion. eLife 4, e11205.

Category