From OpenWetWare
Jump to navigationJump to search

Protein Prep Procedure for mbl-short

• Obtain a agarose/amp plate and let it sit at room temp
• Obtain S.O.C. and let thaw at room temp
• Thaw Rosetta Cells and plasmid on ice
• In a .5mL eppendorf tube add 50uL of chloramphenicol and 100uL of water. Spread on plate (with beads)
• Add 2uL of plasmid into the Rosetta cells
• Mix and incubate Rosetta cells with the plasmid in them on ice for five minutes
• Heat shock the cells + plasmid at 42C for 30 seconds
• Place on ice for two minutes
• Add 250uL of S.O.C.
• Plate anywhere from 100-200uL of cells + plasmid onto plate (Spread on plate with beads)
• Let incubate upside down at 37C overnight
• In the morning put in 4C fridge until ready to use later that day

1. Obtain 1L of LB, add 1000uL of Amp.
2. Start a 50mL starter culture add 50ul of chloramphenicol in it, along with a colony or two from your fresh transformation.
3. Put in 37C incubator/shaker overnight
4. The next morning →
i) Pre-warm your LB/amp in 37C shaker for 30min
Take .5mL of 50% Glycerol and .5mL of your overnight starter culture, put in 1.5mL eppendorf tube, mix and store in -80C
iii) Pour starter culture into the 1L of pre-warmed LB
5. Check absorbance every hour until it has reached 1.0 or above.
6. Induce with IPTG so that final concentration is .4mM IPTG
7. After Protein has been induced, let it shake in a 30C incubator for 3 hours
8. Pellet Cells out by spinning them at 6000rpm for 15 minutes
9. Store pellets in a 50mL falcon tube
10. The next morning→Make 500mL of Lysis Buffer, Wash Buffer A and Wash Buffer B

Lysis Buffer-----500mM NaCl, 25mM Tris pH 8.0, 5% Glycerol, 10mM BME
Wash Buffer A--300mM NaCl, 25mM Tris pH 8.0, 0.1% Triton, 5mM BME
Wash Buffer B---1M NaCl, 25mM Tris pH 8.0, 0.1% Triton, 5mM BME
Elution Buffer----10mM reduced glutathione, 150mM NaCl, 50mM Trish pH 8.0, 0.1% Triton, 5mM BME


11. Re-suspend Pellets from yesterday in 50mL of Lysis Buffer.
12. Lyse 6 times with 15 second intervals (Make sure to keep on ice at all times)
13. Spin down for 30 minutes at 12,000rpm
14. Get two 50mL eppendorf tubes with ~ 5-10ml of beads (one for mbl-GST and the other for mbl with no GST tags)
15. Put half of your protein in one falcon tube and half of your protein in the other falcon tube
16. Shake protein in beads, and let rotate in 4C fridge for 1 hour
17. Wash both protein batches 3 times with Buffer B and 2 times with Wash Buffer A
Washing means: Put 10-25mL of Wash Buffer with protein and GST beads, mix, let rotate in 4C fridge for five minutes. Then centrifuge ~300rcg for 2 minutes. Take out supernatant (which is waste) and do another round of washing
18. In one 50mL falcon tube with beads and protein in it, add 10mL of Wash Buffer A (the one with less salt) and 3 tubes (30uL each) of Precision Protease. Mix, and leave rotating at 4C overnight
19. Elute the protein from the other falcon tube by putting in 7mL of Elution Buffer, mixing, centrifuging, collecting supernatant (save because that is where protein nis) and then add 3 more mLs of Elution Buffer, mix,, and centrifuge. Collect supernatant and store supernatant in 4C
20. Put protein through Q Column→

i) Make sure that there is enough Q Column Buffers
Q Column Buffer A….50mM NaCl, 10mM Tris pH 7.5, 1mM EDTA pH 7.5, 5mM BME
Q Column Buffer A w/o salt…10mM Tris pH 7.5, 1mM EDTA, 5mM BME
Q Column Buffer B….1M NaCl, 10mM Tris pH 7.5, 1mM EDTA pH 7.5, 5mM BME
ii) Check to make sure the pumps are working correctly (water should be coming out of them when you open them. If not, then use the syringe to pull water out)
iii) Do the mbl-GST protein first. Filter Protein. Put mbl-GST in (which should be ~10mL) in 40mL of Q Column Buffer A w/o salt. Mix, and put in 4C fridge
iv) Pump wash purify with lines A and B in filtered water. Do this twice
v) Pump wash purify with line A in Q Column Buffer A (with salt) and line B in Q Column Buffer B. Do once
vi) Wash sample loop and and put in protein, filling top part with water
vii) Pick Program →I think it says MonoQ mbl and make sure you change the injection volume to be whatever you need it to be. Nothing else was changed
viii) Enter all data (eluents and protein) and a name you can remember it as
ix) Put in clean fraction tubes and make sure everything is set
x) Click on start and let the program start
xi) Collect initial flow through and anything else you think you should collect
xii) Wait for Atka to finish then put mbl with no GST through Atka. Spin down mbl with no GST and take supernatant.
xiii) Filter protein. Put mbl with no GST in 40mL of Q Column Buffer A w/o salt.
xiv) Follow same procedure for mbl with GST

21. Concentrate Protein→ protein is at 60mM NaCl make it 300mM NaCl
i) Find the right size nitrogen concentrator and rinse off
ii) Concentrate mbl-GST and mbl w/o GST separately
iii) First concentrate mbl-GST and put in a 10,000 MWCO filter. Remember, the shiny side of filter should be facing up
iv) Put rubber ring around filter, adjust the concentrator and add protein
v) Hook up to lid and waste and waste (save waste just in case)
vi) Turn on nitrogen by turning the right knob on first (counter clockwise) and then the left most knob on (counter clockwise)
vii) Make sure pressure is not above 75 psi (usually you want to keep at 60 psi) if pressure is above adjust using middle knob
viii) Stop when amount of protein is between 1-2 ml
ix) Do the same exact thing with mbl w/o GST and make sure to clean out concentrator with water. Use a 1000 MWCO filter

22. Dialyze Protein overnight→
i) Make Dialysis Buffer…500mM NaCl, 25mM Tris pH 7.5, 50% Glycerol, 5mM BME *ADD BME RIGHT BEFORE YOU ARE READY TO DIALYZE*