Denaturing in vivo ubiquitination assay

Denaturing His IP

Denaturing Buffer 

PBS 

10mM imidazole 

6M guanidinium-HCl 

Wash Buffer 1 

PBS 

25mM imidazole 

6M guanidinium-HCl 

Wash Buffer 2 

PBS 

25mM imidazole 

0.2% Triton X-100 

Elution Buffer

PBS

250mM imidazole 

1 10cm plate per condition, batch purification

1. Collect cells with PBS and cell scraper. Spin 15min 600 x g.
2. Transfer cells to 1.5mL tube, repeat spin
3. Add 50uL PBS to cells. Mix for homogenous solution (~150uL). Pulse vortex ~10x to resuspend cells (monitor cell pellet)
4. Save 10uL cell suspension as input. Add to 60uL 2x Laemmli buffer + BME. Vortex. Boil for 5 min. Vortex. Store -80C.
5. Lyse cells in 800uL Denaturing Buffer. Use wide bore pipet tip to resuspend
6. Sonicate 5 cycles (20s ON, 40s OFF, 19% amplitude total: 1m40s)
7. Spin lysate full speed for 10min at RT.
8. Equilibrate 50uL HisPur slurry with 600uL Denaturing Buffer. Resuspend the resin and then spin for 2min at 700 x g.
   -HisPur resin doesn’t pellet well with GuHCl-containing buffer. Spin for 1 min, then rotate tube 180° and spin another 1 min.
9. Add cleared lysate to resin. Rotate end-over-end for 2-3 hr at RT.
10. Add 500uL denaturing buffer prior to spin so that resin doesn’t dry
11. Wash the resin with 1.4 mL Wash Buffer 1 with end-over-end rotation for 5min at RT. Spin 2min at 700 x g. Repeat for a total of 2 washes.
12. Wash the resin 2x with Wash Buffer 2 to remove excess guanidinium-HCl.
13. Elute bound His-tagged proteins using one resin bed volume with 50uL His Elution Buffer. Shake at RT for 15min. Centrifuge for 2min at 700 x g. Collect supernatant as eluate.
    -ensure all resin is resuspended during elution
14. Save 45uL eluate

Notes: Avoid the use of EDTA or strong reducing agents (e.g. DTT, BME) which will disrupt the function of the nickel resin. Sodium phosphate buffers recommended for Ni resins.
 

Denaturing cell lysis for IP

• start with 10 cm dishes
• detach transfected cells with cell scraper in PBS
• transfer cells to 15mL Falcon. Centrifuge 600 x g for 10min.
• transfer to 1.5mL epp tube with wide bore pipet tip
• pellet cells in 1.5 ml eppendorf tube (5 min 600 x g)
• remove supernatant. Cells can be flash frozen in liquid N2 at this point.
• measure pelleted cell volume. Add 50uL PBS to generate suspension.
• lyse cell pellet directly in 200 µl 2% SDS lysis buffer (with 0.5mM DTT).  4ml 2%SDS
  • Eject lysis buffer near bottom of tube to displace cell pellet with wide bore pipet. Briefly vortex to homogenize sample
• 10 min 95ºC
• add 600 µl 1% SDS Buffer
• sonicate to disrupt DNA (microtip sonicator). 20s ON, 40s OFF/17% amplitude/5 cycles. (total on time is 1m 40s)
  • Make sure sonicator tip is high enough in sample to lyse membranes but low enough so sample doesn’t foam
• 10 min max. speed room temperature to pellet debris. Make sure not to transfer any of the pellet!
• Equilibrate 22uL Streptactin slurry 1x in 2mL Epp tube with RIPA buffer. Remove supernatant. Add 1000uL 1% SDS buffer (with 0.5mM DTT)
• keep aliquot of lysate as input control (e.g. 40 µl)
• transfer supernatant to beads, discard pellet
• Room temperature IP for 2 hours
• Spin beads + sample at 600 x g for 2 min
• Wash Strep beads 4x with RT RIPA buffer
• After final wash, add 35uL 1x Strep elution buffer. Shake for 30min at RT.