Standard Operating Procedure
Hazards:
Only if youÕre creative.
Protection:
An alert and conscious mind.
Waste:
drain
Spill Clean-Up:
Procedure:
Resuspending peptide
Perform resuspension over sink and change gloves frequently to avoid peptide contamination.
Resuspend in H20 to ~5-6mg/mL. Adjust pH to 8-10 to solubilize peptide, but avoid pH > 10 to prevent peptide hydrolysis.
Folding heavy chain with b2M and peptide
1) Prepare fresh refolding solution:
200mL:
100 mM Tris (2.4g Tris base)
400 mM L-Arginine (16.8g)
2 mM EDTA w/ Tris (0.8mL 0.5M EDTA 100mM Tris)
0.5 mM oxidized glutathione (0.0612g)
5 mM reduced glutathione (3g)
pH to 8.0 w/ 1M NaOH
2) Add protease inhibitors :
2mL PMSF (100mM stock)
100uL pepstatin (2mg/mL stock) {Note: [] may be 1mg/mL, check tube}
100uL leupeptin (2mg/mL stock)
Note: Protease inhibitors are used primarily to protect heavy chain and b2M from proteases in bacterial lysate prep. The BirA recognition motif in the heavy chain is particular sensitive to degradation.
3) Add ~12mg peptide to 200mL refolding solution and cool on ice.
4) Prepare heavy chain and b2M stocks (located in Louie Š80C 2nd shelf 4th rack box A)
a. Heavy chain (H2Kb) 18.6mg (final [] ~3uM) (stock ~40mg/mL)
b. b2M 13.2mg (final [] ~6uM) (stock ~50mg/mL)
c. Divide heavy chain into 3 x 6.2mg samples (155uL in each of 3 tubes)
d. Divide b2M into 3 x 4.4mg samples (88uL in each of 3 tubes)
e. Add 500uL of guanidine solÕn to each tube (Note: Mix guanidine solÕn before adding).
f. Place stocks in Š20C until ready to inject.
5) Place in cold room and begin stirring (vortex should be as deep as possible w/o touching stir bar)
6) Add 1 tube of b2M to refolding solution with 1mL syringe through a 26.5 gauge needle (~2 drops/sec).
7) Add 1 tube of heavy chain to refolding solÕn dropwise (~2 drops/sec) with 26.5 gauge needle.
Note: Always add b2M first before adding heavy chain. Oxidized glutathione is sensitive to light and oxygen content, therefore always cover refolding solution in dark and make sure that the stirring bar is at a setting where it is not creating bubbles.
8) Reduce stirring speed to a minimum and place at 4C in dark for 6-8hrs.
9) Repeat 5)-7) with 2nd aliquots of b2M and heavy chain.
10) Reduce stirring speed; place at 4C in dark overnight (6-12hrs).
11) Repeat 5)-7) with 3rd aliquots of b2M and heavy chain.
12) Reduce stirring speed; place at 4C in dark for ~24hrs.
1) Heat beaker of H2O to boiling.
2) Boil dialysis membrane (MWCO 6000-8000) for ~5 min.
Note: Dialysis membrane needs to be ~14-16 inches in length to accommodate ~250mL of solution. Volume of refolding solution will expand by ~50mL during dialysis. Make sure both sides of membrane are immersed in H20 and not stuck to each other. Two pieces of membrane will separate upon boiling. DonÕt try to separate when membrane is dry; itÕll crack.
3) Fill 4L beaker w/ ~3500mL H2O.
4) Rinse membrane with deionized H2O from faucet on sink, then place in beaker.
5) Clamp dialysis membrane at one end with 2 closures.
Note: Fold membrane in half along width, then fold over lengthwise in about 3 inch segments. Clamp over folds; make sure to leave enough space for extra 50mL.
6) Pour in refolding solution.
7) Clamp other end with 2 closures.
8) Place dialysis membrane w/ refolding solution into 4L beaker w/ H2O. Dialysis tubing will float, so unnecessary to use anything to hold it in place.
9) Dialyse by stirring gently for ~4-6 hrs.
Note: If stirring bar spinning too quickly, the force of the vortex will break the dialysis membrane. Make sure setting is low.
10) Replace with another beaker of H2O. Dialyse for another ~12-16hrs.
Note: Dialysis is necessary for desalting the solution in preparation for passing through an ion exchange column. Removing the arginine also makes the subsequent concentration step go much faster.
Equilibrate column. Superdex 200 HR 10/30 (24mL bed volume). Run 2X bed volume (~50mL) of FPLC buffer through column at 0.5mL/min.
Note: This will take ~2hrs. Concentrate monomers in the meantime.
1) Transfer dialyzed folded
monomers to 50mL conicals. (Keep on ice).
2) Spin down ~3000xg for 5-10min.
3) Filter solution through 0.22um filter.
4) Wash out surfactant in Amicon Ultra centrifuge filters with H2O.
5) Concentrate to ~500uL using Amicon Ultra-15 centrifuge filters (10,000 MWCO)
Turn on UV lamp.
Equilibrate column. Superdex 200 10/30 (24mL bed volume). Run 2X bed volume (~50mL) of FPLC buffer through column 0.5mL/min. Max back pressure of column is 1.5MPa.
Note: This will take ~2hrs. Start column equilibration early in the morning and concentrate monomers in the meantime.
******1) Filter sample (0.22um).******
2) Load 1/2 of sample (~250uL)
Note: Wash needle with buffer and use new syringe. Sample loop should be filled w/ buffer. When removing needle, hold the waste outlet tubing above level of needle so that buffer doesnÕt drain out of sample loop.
3) Check column flow rate is at 0.5mL/min.
4) Transfer column outlet tubing to fraction collector.
5) Start fraction collector.
6) Inject sample.
7) Collect 24 fractions (1mL/ fraction ~50 min run time).
8) Repeat 2)-7).
9) Wash column w/ 2x bed volumes of FPLC buffer.
Note: Folded monomers are usually contained in fractions 13-17.
10) Pool monomer fractions and add protease inhibitors.
0.5uL leupeptin per 1mL (stock 2mg/mL in H2O)
0.5uL pepstatin per 1mL (stock 2mg/mL in DMSO).
Note: Pepstatin may only be 1mg/mL; double check [] listed on tube.
9) Concentrate monomer fractions to ~500uL in Amicon Ultra-4. (Note: Wash out surfactant in Amicon Ultra membrane with H2O before use. Concentrating usually takes 10-20min spin at ~3000xg).
10) Bring volume up to 900uL with H2O or FPLC buffer.
1) Add to 900uL folded monomers the following:
125uL Solution A
125uL Solution B
100uL d-Biotin
10uL BirA enzyme
2) Incubate overnight at RT.
******Note: DonÕt forget to filter sample before loading onto column.******
Follow the same procedure as above, except
Add the following to the pooled fractions:
0.5uL leupeptin per 1mL (stock: 2mg/mL in H2O)
0.5uL pepstatin per 1mL (stock: 2mg/mL in DMSO)
2uL NaEDTA/Tris per 1mL (stock: 0.5M EDTA, 100mL Tris)
Concentrate to ~500uL using 4mL Amicon Ultra-4. (Wash out surfactant before using.)
1) Make standards of BSA. Expected yield of monomers is ~0.5mg-1mg. Therefore prepare standards of 2, 1, 0.5, 0.25 mg/mL.
2) Add 500uL of staining reagent to each standard; also add staining reagent to 5uL and 2.5 uL aliquots of monomers.
3) Incubate for 30min at 37C.
4) Determine OD562 and calculate protein [] referring to standard curve.
5) Bring up volume of monomer prep for final concentration of 1mg/mL with FPLC buffer.