Western blotting protocols for detection of PERK-P T980 and eIF2a-P S51
In vitro (HEK 293 or N2A cells)
- Lyse cell pellets in RIPA buffer directly in well (200-300µL for a 6 well plate well)
- Optional: sonicate for 2x5 seconds at low power (20-25%)
- Centrifuge lysates for 10 minutes at >13000rpm in a cooled benchtop centrifuge
- Measure protein concentrations using BCA assay or Bradford assay
- Make up stock solutions with 4X SDS loading buffer (Biorad #1610747)
- Load 10-30µg into a 10% polyacrylamide gel (we use Biorads miniprotean western blotting system)
- Run gels at ~125V (constant voltage mode) for ~1hr30 mins (until dye front leaves gel
- 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)
- Block in TBS-T with 5% bovine serum albumin (BSA) for 1 hour at room temperature with gentle agitation on a shaking platform
- 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
- Rinse three times with TBS-T before three 10 minute washes in TBS-T at room temperature with gentle agitation on a shaking platform
- 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
- Rinse three times with TBS-T before four 10 minute washes in TBS-T at room temperature with gentle agitation on a shaking platform
- 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)
- Lyse hippocampus in 300µL of RIPA buffer using pellet pestle, leave on ice for 30 minutes (vortex every 10 minutes)
- Centrifuge lysates for 10 minutes at >13000rpm in a cooled benchtop centrifuge to remove insoluble material
- Measure protein concentrations using BCA assay or Bradford assay
- Make up stock solutions with 4X SDS loading buffer (Biorad #1610747)
- 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)
- Run gels at ~125V (constant voltage mode) for around 1hr30 mins (until dye front leaves gel
- Wet transfer at 20V overnight (or at least 16hrs) onto 0.45µm PVDF membrane (we use IPVH00010 from Merck)
- Block in TBS-T with 5% bovine serum albumin (BSA) for 1 hour at room temperature with gentle agitation on a shaking platform
- 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
- Rinse three times with TBS-T before three 10 minute washes in TBS-T at room temperature with gentle agitation on a shaking platform
- 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
- Rinse three times with TBS-T before four 10 minute washes in TBS-T at room temperature with gentle agitation on a shaking platform
- 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