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Buffer Exchange and Solution Concentration by hcj

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									Buffer Exchange and Solution Concentration

Prep Work
   1. Take UV-Vis absorbance reading of stock sample and determine starting concentration

   2. Place 450µL of stock sample in 1.5mL sample tube and add 50µL 100% D2O (NOTE: This gives a 10%
      D2O solution for an NMR lock, if a different concentration is desired adjust accordingly)

   3. Transfer sample to NMR tube and run an HSQC on the 500MHz NMR to check stock sample stability
      (NOTE: See 500MHz HSQC protocol)

   4. If stock sample looks good move on to buffer exchange (NOTE: Because the buffer exchange is time
      consuming it is beneficial to plan ahead! Determine variables and prep buffer exchange solutions before
      hand!)

   5. If sample does not look good, determine/fix problem(s) and repeat steps 1-3 until sample is satisfactory

Buffer Exchange
   1. Place 500µL of stock sample on the CENTRIPREP column

   2. Add 1000µL of exchange buffer to the column

   3. Attach elute collection tube to bottom of column and place the cap on top

   4. Place in centrifuge and spin down for ~20-30min at 5,000rpm (NOTE: The goal is to decrease the total
      volume back to original volume (~500µL), depending on your sample you may need to increase/decrease
      centrifuge time. DO NOT vary centrifuge speed this may cause clogging of column pores or worse!)

   5. Take sample out of centrifuge and add another 500µL of exchange buffer

   6. Repeat steps 2-5 about 4 times or until buffer exchange is complete

   7. After exchange is complete remove sample from column and place in 1.5µL sample tube

   8. Take a UV-Vis absorbance reading of sample using a 100X dilution to check final concentration (NOTE:
      100x dilution is 990µL of exchange buffer and 10µL of sample)

   9. Check the % recovery using equation 1

                                           (Finalvolu me)(FinalA BS)
                             %recovery=                                 (100 %)   [1]
                                          (Initialvo lume)(Init ialABS)

   10. Remove 450µL of sample and place in new 1.5µL sample tube and add 50µL 100% D2O. (NOTE: This
       gives a 10% D2O solution for an NMR lock, if a different concentration is desired adjust accordingly)

   11. Transfer sample to NMR tube and run an HSQC on the 500MHz NMR to check stock sample stability
       (NOTE: See 500MHz HSQC protocol)

   12. If buffer exchange is used to check long term stability at room temp, store sample(s) at room temperature
       for the desired length of time while periodically checking stability using HSQC and note any resulting
       changes.
Solution Concentration
After optimal buffer conditions are determined the sample is ready to be concentrated. However, by increasing the
concentration of protein there is the chance that optimal buffer conditions will change also. Thus further
optimization will be needed.
    1. Run a number of buffer exchanges (~4) at the experimentally determined optimal conditions. (NOTE: You
       should collect ~2mL of sample for concentration. Collect and combine each exchange sample in a 2mL
       sample tube)

   2. Check UV-Vis absorbance and run an HSQC to prove sample is soluble and stable

   3. If sample is soluble and stable than continue with concentration. (NOTE: If sample is not soluble, stable, or
      both determine/fix problem(s) and start over).

   4. Place 1mL of sample on the CENTRIPREP column

   5. Attach elute collection tube to bottom of column and place the cap on top

   6.    Place in centrifuge and spin down for ~20-30min at 5,000rpm (NOTE: The goal is to decrease the total
        volume back to original volume or about 500µL so depending on sample may need to increase/decrease
        centrifuge time. DO NOT vary centrifuge speed this may cause clogging of column pores or worse!)

   7. Remove 10µL of concentrated sample and place in 990µL of optimal exchange buffer (NOTE: 100X
      dilution for UV-Vis)

   8. Check UV-Vis absorbance and run an HSQC to prove sample is soluble and stable

   9. If everything worked well and you have reached the desired sample concentration with relatively few
      problems such as aggregation or precipitation, consider yourself lucky and prepare for analysis using the
      600MHz NMR!

								
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