First, the buffer was prepared by using the formula as follows: Figure 1: Calculation for prepare 0. 1 M potassium phosphate buffer at pH 6 3. 4007g of potassium phosphate was weighed and placed in 300 ml beaker. Then, 125 mL of water was added into the beaker that contained potassium phosphate. The mixture was dissolved using the stirring rod, and then the magnetic stirring bar was placed in the beaker for further dissolve when measuring the pH. The pH meter was used to measure the solution, and the data was documented at pH 4. 6. This was the starting point. Next, 1M NaOH was slowly added into the buffer to make it to pH 6.
Then, the buffer was transferred to a 1000 ml graduated cylinder, and 125mL of distill water was added to the buffer. Next, the buffer was transferred to 250 mL glass jar and was labeled 0. 1M Potassium Phosphate buffer, pH 6. Other group was prepared other buffers that have a different pH (pH 4,8,and 10). They are 0. 1 M Sodium acetate buffer at pH 4, 0. 1 M Sodium phosphate buffer at pH 8,0. 1 M Glycine buffer at pH 10. The packed gel-chromatography column was obtained and was clamped to a ring stand (the column was prepared in for lab 2, see experiment 2 for how to pack the column).
Then, 50 mL total of 0. 1M Potassium Phosphate buffer pH 6 was measured by the 50 mL graduated cylinder and was transferred in the column in order to equilibrate the column. 5ml of the buffer was measured each time by the by the P-1000 micropipettes and transferred in the column slowly on the side of the column in order to prevent overflow and prevent the bed from disturb. Make sure that not to let the buffer level fall below the bed. The column was stopped when there is about 1 cm of the buffer on the top of the bed.
The beaker was placed at the bottom of the column to collect the buffer that runs through the column. While waiting for the buffer run through the column, 1% Phenol Red was prepared by using the formula as follows: Figure 2: Calculation for preparing 1% phenol red 0. 01 g of phenol red was weighed and was placed in the test tube. 1mL of buffer (0. 1M potassium phosphate, pH 6) was measured by the micropipette and was added into the test tube that contained the phenol red. The yellow color was presented indicating it is acidic. Everything was stored at 2-8C for the second day experiment.
Day 2: The column was obtained and was clamped to the ring stand, then the level of buffer about 1cm above the bed was allowed to run through the column until it reach the surface of the bed. Once again, very careful not to let the bed run dry because it will cause uneven flow of the solutes. Next, 17 clean, dry cuvettes were obtained and labeled 0 to 16. The microcentrifuge tube was obtained and labeled ‘loaded sample’. 0. 3 mL of 1% phenol red was measured and transferred to the microcentrifuge tube by using the micropipette p-1000.
Then, 0. ml buffer was measured and added into the microcentrifuge tube that contained phenol red. 0. 020g Bovine Serum Albumin (BSA) was weighed and was placed in the microcentrifuge tube that had the phenol red and the buffer. The mixture was gently mixed. Shaking will cause denature of the protein. The next step was making the blank. 1mL of buffer was measured and transferred to the cuvette that labeled 0 (blank). After the blank was made, 250ul of the mixture (loaded sample) was measured and loaded into the column by the P-1000 micropipette. The mixture was put gently on the wall of the column in order to not disturb the bed.
As soon as the color of solution entered the bed, 3mL of buffer was added to the column and was allowed to run through the column. The buffer was added continuously to the top of the column. There was 19 mL of buffer in total. 1mL fraction was collected in the each cuvette in order corresponding to the labels. The fractions were collected into each cuvette until there is no color present in the last fraction. 1 mL of fraction was measured by keeping the blank cuvette that contained 1ml of buffer next to the cuvette so you know when the fraction is reach 1mL.
When the last fraction that obtained was colorless. 250 uL of 1M NaOH solutions was measured and added in each cuvette contained the fraction, including th blank. The purpose of adding NaOH is to change the color in every group to the same color in order to read at the same wavelength (520 nm). LIE Figure 3: Picture of column that shows the separation of albumin-phenol red complex and free phenol red by gel chromatography. Figure 4: Picture of sample fractions that collected using gel chromatography. The more color the more concentration. The color of fractions turned from orange to purple-red after adding he 250 uL of 1M NaOH. All the cuvettes that contained the fraction were run through the Spectrometer #2 that set to 520 nm to measure the absorbance. Before measuring the fractions, the blank cuvette that contained 250 uL of 1M NaOH solutions and 1 mL of buffer was placed in the machine and was pressed “auto zero”. Then, each of cuvette that contained fractions was placed in the machine and the absorbance was read. All the data were collected and recorded in table 1. All the waste was discarded to the appropriate waste container. Next, the data were entere in Excel for graphing.
The ‘fraction number and the ‘absorbance number’ were used to construct the graph. Select the ‘Fraction number’ row for X-axis and then selects the ‘Absorbance’ row for Y-axis. Then, go to Chart – Marked Scatter – Smooth marked scatter to build the graph. Next step is go to chart layout and input the chart title, axis title. After the graph was constructed, the percent bound was calculated for pH4, pH6, pH8, and pH10 (the data for pH 4, 8, 10 were given from other group) based on the formula: % Bound= (max of peak 1/ (max of peak 1+ peak 2)) x 100 For pH 4, peak 1 is 0. 99 and peak 2 is 3. 215. There was another peak at absorbance 0. 502, however, it is too small compare to 2 other peaks that we have (Figure 6), so the 2 peaks should be 0. 199 and 3. 215. % Bound =((3. 311/ (3. 311+2. 069)) x 100 = 61. 543 % The way of choosing which one is peak 1 and peak 2 for pH 6, pH 8, and pH 10 was done the same as pH 4. Then, the percent bound of pH 6, 8, 10 were calculated using the same formula as above. The data were documented in table 1. Next, the percent bound graph was constructed using the pH and percent bound data that calculated.
The data were entered in Excel, then select the pH row for ‘x-axis’, and select the ‘percent bound (%) row for y-axis. Next, go to Chart – Marked Scatter > Smooth marked scatter to build the graph. Then, go to chart layout and input the chart title, axis title. Data and Results: Table 1: Absorbance measured for each fraction at 520nm. The Fractions were collected using the buffer pH 6, pH 4, pH 8, and pH 10. The percent bound of bovine serum albumin and phenol red at different pH.
Fraction Number Absorbance @520 nm pH 6 pH 4 pH 8 pH 10 00. 000 0. 000 0. 000 0. 00 10. 110 0. 502 0. 002 0. 005 20. 060 0. 146 0. 021 0. 000 30. 119 0. 169 0. 079 0. 076 40. 078 0. 232 0. 044 0. 006 50. 157 0. 383 0. 062 0. 006 60. 713 1. 000 0. 093 0. 030 72. 683 2. 395 0. 218 0. 246 83. 215 3. 215 1. 654 1. 922 93. 215 3. 311 3. 311 2. 709 10 3. 215 3. 215 3. 311 3. 215 11 2. 088 2. 075 3. 215 3. 215 12 0. 370 2. 069 3. 135 3. 215 13 0. 091 0. 880 2. 914 3. 215 14 0. 043 0. 353 0. 916 2. 079 15 0. 032 0. 279 0. 888 16 0. 029 0. 109 0. 216 17 0. 085 0. 063 18 0. 063 0. 021 0. 041 0. 032 20 0. 050 21 0. 050 22 0. 025 23 0. 021 Peak 1 0. 19 3. 311 0. 079 0. 076 Peak 2 3. 215 2. 069 3. 311 3. 215 Percent bound (%)3. 569 61. 543 2. 330 2. 309 Figure 5: The plot of Fraction number vs. Absorbance that measured at 520 nm. The fractions were collected using the buffer pH 6. Figure 6: The percent bound of bovine serum albumin and phenol red at different pH.
Discussion: The pH that has optimal binding was expected is pH 4. As you can see in table 1, the percent bound of BSA-PR at pH 4 (61. 543%) was highest compare to other pH. pH 6 has the percent bound of 3. 569%, pH 8 has the percent bound of 2. 30%, and pH 10 has the percent bound of 2. 309%. pH 6, 8, and 10 do not have an optimal binding for bovine serum albumin and phenol red because they have very low percent bound (refer to table 1), mostly is the free phenol red. Therefore it matches with the prediction. Moreover, there were 2 peaks presented when graphing. Based on the order of elution, the first peak is the bound complex of BSA-PR. According to the data in table 1, the percent bound of BSA-PR was 61. 65%. It is not 100% bound, so the second peak is the free phenol red.
It is the same with pH6, pH8, and pH 10. Order of elution based on the size of the molecules, the complex of BSA-PR (68,000 Da) eluted first because its size is bigger than the exclusion size of the resin and cannot enter the pores of the bead. Thus, it will travel rapidly and elute first. The BSA is about 66,000 Da, and the phenol red is about 350 Da. The free phenol red will elute last due to its smaller size (350 Da). Small molecules easily enter and fit in the many pores of the beads. Therefore, it takes more time to run down the column and then elute slower.