(As an alternative to disuccinimidyl suberate (DSS) crosslinking)
Reagents:
- PBS
- Binding buffer: 0.2M sodium borate (adjust 0.2M Boric Acid to pH 9 with NaOH)
- Crosslinking reagent: 20mM DMP (dimethylpimelimidate, Pierce # 21666) dissolved in 0.2M sodium borate – make immediately prior to use
- “Quenching” reagent: 0.2M ethanolamine (pH 8.0 in 50 mM Ammonium Bicarbonate)
- Acid Wash buffer: 0.58% v/v acetic acid with 150mM NaCl
- Lower stringency lysis buffer for wash: 150mM NaCl, 50mM Tris, 10mM EGTA, 0.2% NP40
- MS Wash buffer: 50mM ammonium bicarbonate
- Elution buffer: 0.25% Rapigest SF Surfactant in 50mM ammonium bicarbonate
Procedure:
Coupling antibody with agarose beads:
- Couple antibody/control IgG andProtein A/G beads (Pierce # 20421) in PBS:
- Add 60ul (20 ug) X antibody to 30 ul Prot A/G beads in 1ml PBS
- Add 50ul (20 ug) IgG rabbit to 30 ul Prot A/G beads in 1ml PBS (control)
- Rock 2-3 hours or overnight at 4C
- Wash beads 3X with 1ml of 0.2M sodium borate pH 9 – after each wash, spin at 300-500g to pellet beads, gently remove supernatant
- Save small sample to run on gel to check efficiency (see step 15)
Crosslinking:
- Make fresh DMP:
- Let solid warm up to room temperature ~20min
- Weigh out 0.0259 g DMP
- Immediately add DMP to 5ml of 0.2M sodium borate (pH 9) to make 20mM DMP solution
- Immediately add 1ml 0.2M sodium borate + 20mM DMP to (1) beads + X Antibody; and (2) beads + Control IgG
- Rock at RT for 40min to perform antibody crosslinking
- Spin down and remove supernatant
- Wash beads once in 0.2M ethanolamine (pH 8.0) – this removes/quenches any residual DMP
- Resuspend in 1ml of 0.2M ethanolamine (pH 8.0)
- Rock at RT for 1-2 hours
- Save small sample to run on gel to check efficiency (see step 15)
- To remove uncoupled IgGs, wash with 3X 1mL 0.58% v/v acetic acid + 150mM NaCl
- Wash 3X with 1ml cold PBS - after each wash, spin at 300-500g to pellet beads, gently remove supernatant
- Save small sample to run on gel to check efficiency (see step 15)
Check the efficiency of immobilization:
- Sample beads (1) before and (2) after crosslinking, and (3) after the acid wash – elute by boiling resin in 2x LDS loading buffer, then run SDS-PAGE/Coomassie stain . **Ensure equal volumes of beads are analyzed for each of these samples. For example, if you are going to take out 5 uL of beads for the check and then elute in 20 uL 2X LDS and load 10 uL for SDSPAGE, be sure to do it the same for all resin binding steps.
- Evaluate the results of the immobilization process prior to proceeding. (please share images with us!!)
Proceed with IP:
- Quantity of input material will vary depending on the expression of the target protein/complex. Typically, between 0.5 and 5 mg input protein is utilized. Preclearing is recommended, by adding your lysate to ~25 uL of uncoupled Protein A/G resin, rock for 30 minutes, and reserve supernatant for IP.
- Add 1ml (~1-5 mg/mL protein) of precleared lysate to approximately 25 uL coupled resin. Use same resin quantity for all samples.
- Rock overnight at 4C (with end-over-end mixing, if possible)
- Save supernatant for WB analysis.
- Wash resin 3X with lysis buffer containing 150mM NaCl, 50mM Tris, 10mM EGTA, 0.2% NP40 (you may want to save these washes for troubleshooting purposes… a signal in WB at this step (but not step 20) would suggest target bound to antibody but was then washed away)
- Elute with 50-100ul of 2X LDS loading buffer, boil 5 min at 95C, pellet beads by centrifugation, take supernatant for SDSPAGE analysis (below). ***see step 23 for MS compatible elution protocol
Recommended Setup for Coomassie gel (or WB analysis):
- Ladder
- 50ug lysate
- X Beads pre-elution
- X Beads post-elution
- X Elution supernatant
- IgG Rab Beads pre-elution
- IgG Rab Beads post-elution
- IgG Rab Elution supernatant
*Stain with Invitrogen Colloidal Blue Staining kit (LC6025)
*Alternative elution steps once IP has been optimized
- After step 21 wash, wash resin 3X with 1 mL 50mM ammonium bicarbonate
-
Add 50-100ul of 0.2% Rapigest SF Surfactant (Waters Corporation) in 50mM Ammonium Bicarbonate.
-
Boil @ 95C for 5min, pellet beads by centrifugation, take supernatant for delivery to Duke Proteomics Core Facility, or for SDS-PAGE analysis as above.