Given this procedure, what would be the total volume of protein mixture in each fraction (1-7)?
400 ul
tube. The cap will not close. Rinse the spin column by loading 50 ul of Wash buffer on top of the membrane. Using a microcentrifuge, spin the column at 3,400 rpm for 5 minutes. Verify that all the buffer solution has passed through the spin column and has collected at the bottom of the microcentrifuge tube. If liquid is still visible above the resin bed in the spin column, centrifuge for an additional 2 minutes.
2. Remove the spin column from the microcentrifuge tube and discard the microcentrifuge tube. Place the spin column into a new 1.5 mL microcentrifuge tube. 400 ul of Dotcnmixtu 3. Transfer 100 ul of protein mixture to a microcentrifuge tube and add 0 ul of wash buffer. Load all of the protein mixture solution onto the spin column and centrifuge the column at 3,400 rpm for 5 minutes collecting the effluent, if any, in the tube. If liquid is present in the collection tube label it Fraction 1.
4. Pull the spin column out of the microcentrifuge collection tube and insert it into a new microcentrifuge tube. Add 400 ul of Wash buffer and centrifuge the column at 3,400 rpm for 5 minutes collecting the effluent in its microcentrifuge tube. Save the solution and label the tube Fraction 2.
5. Repeat step 4 with an additional 400 ul of Wash buffer. Save the solution and label it Fraction 3.
6. Repeat step 4 with an additional 400 ul of Wash buffer and save the solution labeling it as Fraction 4.
7. Load 400 ul of ELUTION BUFFER onto the spin column and centrifuge for 5 minutes at 3,400 rpm collecting the effluent in its microcentrifuge tube. Save the solution and label it Fraction 5.
8. Repeat step 7 two more times with the elution buffer. Save the solutions and label them Fractions 6 and 7.
9. Add 400 uL of the wash buffer to each fraction.
Analysis of Fractions 10 Set up the spectrophotometer by pressing ENTER GQTO WL280. At the end of this sequence, the wavelength should read 280 nm in the upper right-hand corner of the screen. If it does not consult your instructor. Place a UV-compatible cuvette in the cuvette holder. Be sure the cuvette is in the correct orientation.