Western blotting

Western blotting protocols for detection of PERK-P T980 and eIF2a-P S51

In vitro (HEK 293 or N2A cells)

1. Lyse cell pellets in RIPA buffer directly in well (200-300µL for a 6 well plate well)
2. Optional: sonicate for 2x5 seconds at low power (20-25%)
3. Centrifuge lysates for 10 minutes at >13000rpm in a cooled benchtop centrifuge
4. Measure protein concentrations using BCA assay or Bradford assay
5. Make up stock solutions with 4X SDS loading buffer (Biorad #1610747)
6. Load 10-30µg into a 10% polyacrylamide gel (we use Biorads miniprotean western blotting system)
7. Run gels at ~125V (constant voltage mode) for ~1hr30 mins (until dye front leaves gel
8. Wet transfer (again Biorads miniprotean system) for 2 hours at 400mA (constant current mode). Store transfer on ice or in cold room for duration. Use 0.45µm PVDF membrane (we use IPVH00010 from Merck)
9. Block in TBS-T with 5% bovine serum albumin (BSA) for 1 hour at room temperature with gentle agitation on a shaking platform
10. Incubate with antibodies (PERK-P #3179 CST and eIF2a-P #3597) at 1:2000 in TBS-T with 5% BSA overnight at 4°C with gentle agitation on a shaking platform
11. Rinse three times with TBS-T before three 10 minute washes in TBS-T at room temperature with gentle agitation on a shaking platform
12. Incubate with HRP conjugated secondary antibody (we use Biorad #1706515) at 1:5000 in TBS-T with 5% BSA for 1 hour at room temperature with gentle agitation on a shaking platform
13. Rinse three times with TBS-T before four 10 minute washes in TBS-T at room temperature with gentle agitation on a shaking platform
14. Develop using chemiluminescence substrate (we use WBKLS0500 from Merck), we have used both a dark room with old style developer or Biorad Chemidoc

In vivo (mouse hippocampus)

1. Lyse hippocampus in 300µL of RIPA buffer using pellet pestle, leave on ice for 30 minutes (vortex every 10 minutes)
2. Centrifuge lysates for 10 minutes at >13000rpm in a cooled benchtop centrifuge to remove insoluble material
3. Measure protein concentrations using BCA assay or Bradford assay
4. Make up stock solutions with 4X SDS loading buffer (Biorad #1610747)
5. Load 20-40µg into a 10% (consider 8% or even 6% for PERK) polyacrylamide gel (we use Biorads miniprotean western blotting system and cast our own gels)
6. Run gels at ~125V (constant voltage mode) for around 1hr30 mins (until dye front leaves gel
7. Wet transfer at 20V overnight (or at least 16hrs) onto 0.45µm PVDF membrane (we use IPVH00010 from Merck)
8. Block in TBS-T with 5% bovine serum albumin (BSA) for 1 hour at room temperature with gentle agitation on a shaking platform
9. Incubate with antibodies (PERK-P #3179 CST and eIF2a-P #3597) at 1:1000 in TBS-T with 5% BSA for three hours at room temperature with gentle agitation on a shaking platform
10. Rinse three times with TBS-T before three 10 minute washes in TBS-T at room temperature with gentle agitation on a shaking platform
11. Incubate with HRP conjugated secondary antibody (we use Biorad #1706515) at 1:5000 in TBS-T with 5% BSA for 1 hour at room temperature with gentle agitation on a shaking platform
12. Rinse three times with TBS-T before four 10 minute washes in TBS-T at room temperature with gentle agitation on a shaking platform
13. Develop using chemiluminescence substrate (we use WBKLS0500 from Merck), we have used both a dark room with old style developer or Biorad Chemidoc

Recipes

RIPA – 50mM Tris pH 7.5, 150mM NaCl, 1% NP40, 0.5% Na deoxycholate, 3mM EDTA supplemented with cOmplete, Mini, EDTA-free, Sigma-Aldrich; PhosSTOP, Sigma-Aldrich