Concentrating the Protein after Dialysis

 

  1. After the dialysis has finished remove the sample from the cold room and prepare an ice bath.
  2. Save about 15 ml of the dialysis buffer for a blank sample in the spec and for dilution of the protein.  Put 15ml in a small conical tube and place on the ice bath.
  3. Grab a large 50 ml conical tube and label it appropriately.  Carefully remove one of the clamps and transfer your protein into the conical tube and place in the ice bath.
  4. Create a 1mL sample of your protein for the spec making a 1:50 dilution with the extra buffer. (20uL protein and 980uL buffer)  Place the 1ml dilution into an eppendorf tube and place on the ice bath.
  5. Grab the two glass cuvettes and the key for the Brand Lab and go over to the lab with the spec. 
  6. Turn on the spec and allow it to warm up and calibrate. 
  7. Use a 1mL sample of the buffer as a blank, put it in one of the cuvettes, wipe the sides with a Kimwipe, and place in the back holder in the machine.
  8. Load your protein sample into the other glass cuvette, wipe the sides with a Kimwipe, place in the front holder of the machine and close the lid.
  9. Pick the number 2 option that says “Spectrum” and hit <ENTER>
  10. When the screen comes up saying “Change Parameters Y/N” hit the Yes
  11. Choose the first parameter which is wavelength and change the start wavelength to 400nm and the end wavelength to 230nm.
  12. Change the third parameter to have a max of 0.5
  13. Once the spectrum is complete it will display “Data Processing Y/N.”  Hit the yes button and then press 5 to print the data.  Once the data is done printing hit the <COPY> button to print a copy of the spectrum.
  14. Remove the cuvettes from the spectrum and place them in the cuvette cleaner and clean with dH2O and then 95% ethanol.
  15. Leave everything in the lab and leave the spec on so that you can use it again when you are done concentrating your protein, but remember to grab the key and close the door.
  16. Grab one of the special concentrating centrifuge tubes which are over by Chris’s desk.  It should say 5000 on the top as the Molecular Weight Limit of the filter.
  17. Pour your entire sample in and run on the centrifuge at a speed of 3700 for 10 – 12 minutes.  When it is done running check the tube for any liquid and run the centrifuge for a little longer if necessary.
  18. Once all the liquid is gone get the second part of the centrifuge tube to collect your protein.
  19. Then put the special part with the thin needle like side facing up in the centrifuge and run at a speed of 600 for about 5 minutes.
  20. Be sure to save the liquid part from the previous centrifuge just in case there is a problem and your protein is not filtered out.  Keep this liquid on ice.
  21. Once the centrifuge is done remove the protein and place in an eppendorf tube being very careful not to perturb the protein or cause air bubbles.   
  22. Make a 1:200 dilution of your protein with the extra buffer (5uL and 995uL buffer) and place in an eppendorf tube and place on ice.
  23. Do another spectrum so that you can determine the concentration of your protein.  (A = ЄCl where Є = 35410)  *When doing the math be sure to multiply the absorbance by the dilution factor.

24. Once you have determined the concentration label the eppendorf tube and store in a fridge.