June 18, 2014
Solutions that were dialyzed were checked using the spectrophotometer, and it was determined that nearly all protein that was extracted and run through the column was lost during dialysis. Today we are restarting the experiment, and plan to do a few things differently to try to minimize the loss (try manipulating pH, using more dilute myoglobin/buffer solutions, and use different methods/materials for the dialysis).
- 6 g Tris dissolved into 500 mL H2O and titrated to a pH of 7.2
- 50 mg of myoglobin dissolved into 20 mL of buffer twice (to produce 2 solutions)
- 20 mL of butanone measured out twice (equal parts butanone for each of the buffer/myoglobin solutions)
- Ice bucket prepared, buffer/myoglobin buffer solutions and butanone chilled
- Ring stand and filter funnel apparatus set up
- Titration calculations done to figure out volume of acid needed to drop each of the buffer/myoglobin solutions to pH 2.5 and 3 respectively
- This is to see if changing pH will impact extraction process
pH 2.5 Extraction:
- Once solutions have chilled, 1M HCl is added to one of the solutions to make the pH 2.5, new solution is poured into extraction funnel, and butanone added immediately after. Solution is mixed for 30 seconds.
- Three distinct layers formed: upper (butanone/heme, deep reddish brown), middle (clear), and lower (aqueous organic)
- Top layer of butanone/heme was siphoned off using a glass pipette and pipette helper.
- Saved in a Falcon tube for later spectral analysis
- Since more than two distinct layers formed, solution must be treated with butanone again, and mixed for 30 more seconds.
- After a minute, two distinct layers formed: upper (dark red) and lower (clear with a pink tint)
- One more treatment of butanone is done to ensure total extraction, and mixed for 30 seconds
- Two layers formed: upper (clear butanone/heme) lower: (clear but foggy apoprotein layer and Tris buffer)
- Upper layer siphoned off
- pH strips used to test the pH of the clear apoprotein fraction, to ensure that it didn't change too dramatically during the extraction process. Rough readings from strips were unclear, so pH probe was used to determine that the final pH of the solution was 1.14
pH 3.0 Extraction:
- The same procedure described above was used for this extraction
- Exception: pH was far too low in the last experiment, so only 1 mL of 1M HCl was added
- Two distinct layers formed in the first butanone treatment: upper (dark red butanone/heme), and lower (apoprotein and Tris)
- Second butanone treatment produced the previously seen clear upper layer, and the slightly cloudy clear lower layer
- This treatment produced results similar to the third treatment of the previous extraction
- Third butanone treatment was used, and produced highly similar layers to the previous butanone treatment. No notable differences were observed
- Final pH was determined to be 1.42
UV Visible Spectral Analysis of Apoprotein:
- Analysis of the pH 2.5 sample revealed the presence of concentrated protein at the anticipated wavelength (around 280 nm)
- Protein was too concentrated (absorbance above 1 at 280 nm), so a 1/20 dilution was used
- Protein was still too concentrated, so a 1/400 dilution was used
- All initial spec data was saved using data print table format (.txt)
- Edit > method >file name (saved as .spc file, saved within program)
- To start the spec, the green button on the keyboard is pushed
- Naming: Apo_extraction#_pH_TypeOfDilution)
- Analysis of the pH 3.0 sample revealed a similar finding, and the same dilution was used to analyze it
- A sodium bicarbonate (NaHCO3) solution was prepared for dialysis (3.36g NaHCO3 into 4L H2O)
- Apoprotein solutions transferred to dialysis bags, placed in sodium bicarbonate solution, and allowed to rest overnight in the refrigerator.
- The bag with the orange clamps is pH 3, and the bag with the green clamps is pH 2.5