MGH1-binding Protein Enrichment

Solutions and materials: Pure MGH1 powder
Dry NHS-activated agarose beads
1x PBS
DMSO
modRIPA:

• 1% NP-40

• 25% Sodium Deoxycholate

• 150 mM NaCl

• 1 mM EDTA

• 50 mM Tris pH 7.5

• 1x HALT protease inhibitor cocktail, EDTA-free
  
  

Day 1, If using adherent cells, Preparation of Proteins for enrichment
Description: We want fresh protein lysate ideally (never frozen to avoid denaturation).  At least 2mg of total protein is required in a volume of ~1mL -1.5mL. This protocol should wash all proteins from the media away.  If using worms, a similar strategy should work well.

1. Wash adherent cells once with cold PBS (15 mL is cells are in T-175 flask).

2. Add 15 mL of cold PBS to cells.

3. Scrape cells to detach.

4. Triturate and collect cells/PBS in 15 mL tube.

5. Pellet cells 3,000 rcf for 3min at 4 degrees Celsius.

6. Remove PBS with aspirator.

7. Resuspend cell pellet using 1 mL cold PBS, and break up clumps by pipetting with 1mL pipet tip.

8. Add 4 mL additional cold PBS to dissolved cells, and mix by pipetting.

9. Pellet cells 3,000 rcf for 3 min at 4 degrees Celsius.

10. Remove PBS with aspirator.

11. Store washed cell pellet on ice until lysis.

12. Add modRIPA to cells (10 mL buffer per 2x108 cells).

13. Vortex cells to completely dissolve pellet.

14. Sonicate cells on ice at lowest power setting for 3 cycles of 10 seconds each.

15. Clarify protein lysate by centrifugation at 20,000 rcf at room temp.

16. Move supernatant to new tube. Quantify protein using BCA assay.
    
    

Day 2, Preparation of immobilized MG-H1 beads
Description:  For every enrichment, we want to use at least 25 uL (bead volume) containing 3 uMoles of immobilized MGH1 on the surface.  The dry beads swell to about ~7.5 mL/gram, so 7.2mg swelled should produce roughly 50 uL of swelled beads.  The reaction efficiency will positively correlate with the concentration of MGH1 during the reaction, so we want to minimize the volume of MGH1 solution per unit of beads.

1. Weigh 7.2 mg of dry NHS-activated agarose per 2 affinity enrichments.

2. Dissolve 10 mg MGH1 powder at 0.25 M using 88 uL PBS (0.5 uMoles/uL or 0.5 M).

3. Add MGH1 solution containing 6 uMoles (12 uL of the above solution) to NHS-activated agarose beads. 

4. Add 76 uL additions PBS to the beads to create about 50% bead slurry.

5. Allow MGH1 to react with beads for 1 hour at room temp. Mix periodically by tapping the bottom of the tube.

6. Wash beads once with 1 mL PBS to remove unbound MGH1.

7. Repeat step 5 for a total of 2x PBS washes

8. Add 200 uL 1M Tris, pH 7.5 to beads to quench unreacted NHS groups.

9. Allow quench to react for 30 minutes at room temp. Mix periodically by tapping the bottom of the tube.

10. Remove Tris by aspirating

11. Wash beads twice with 1mL PBS.

12. Resuspend beads with PBS and split into 2 tubes. Remove most PBS.

13. Pipet protein solution containing ~2 mg in 1 mL of buffer onto beads.
    

14. To soluble competitor control, add 10X molarity of the small molecule.
    

15. Incubate overnight at 4 degrees Celsius or for 2h at room temp on rocker platform.
    
    

Day 3, Washing/elution of beads and gel running:

1. Wash beads three times with 1 mL modRIPA buffer
   

2. After final wash, completely aspirate all buffer with flat gel-loader tip.
   

3. Add 20 uL modRIPA to beads.
   

4. Reduce with 4.5 mM DTT for 30 minutes at 37 degrees.
   

5. Alkylate with 10 mM for 30 minutes in the dark at room temp.
   

6. Add 1 part 4X LDS sample buffer to 3 parts bead solution and heat beads at 70 degrees Celsius for 10 minutes to elute proteins.
   

7. Remove all elution solution with a flat gel-loading tip and load onto a gel.
   

8. Spin eluted protein solution for 2 minutes at 10k rcf at room temp to pellet any bead carryover.
   

9. Load eluted protein solution on gel with flat gel loader tip and run gel. Stain with spyro ruby using rapid protocol and take a picture using biorad geldock.
   Cut gel into six slices per lane, proceed with in-gel digestion procedure.
   

Day 3: Stage Tip cleanup of peptides from in-gel digestion, 2 hr:
****Spin ~1 minute at 3,000 rcf to force liquid through at each step

1. Make StageTip with 2x 18GA C18 disks in 200 uL pipette tip.
   

2. Wet StageTip with 100 uL of 100% ACN.
   

3. Wash StageTip with 100 uL 50% ACN, 49.8% water, 0.2% FA.
   

4. Equilibrate Stage tip with 100 uL 0.2% FA in water.
   

5. Repeat step 22 once for a total of two 0.2% FA equilibrations.
   

6. Load peptides eluted from IP.
   

7. Wash peptides with 100 uL 0.2% FA in water.
   

8. Repeat step 25 once for a total of two washes.
   

9. In a new tube, elute with 50 uL of 50% ACN, 49.8% Water, 0.2% FA.
   

10. Dry peptides completely in speedvac.
    

11. Resuspend peptides with your favorite injection solution + 0.1 uL HRM standards per injection (e.g. if desired resuspension volume is 10 uL, and 3 uL will go on column, add 0.33 uL HRM standards and 9.67 uL resuspension solution). For single-injection SWATH workflow, add 6.8 uL 3% ACN/0.2%FA + 0.2 uL HRM standards.  Sonicate in water bath 5 minutes.  Vortex at least 5 minutes. Spin at >12,000 rcf for >2 minutes and transfer to autosample vial.