Lab Report #5: Chromatography and Applications
Madison Stargell
Section: BIOL 3520.507
TA: Dennis Carty
Overall objective
Chromatography can be used as a purifying technique by separating individual
components in a mixture. In this lab, we use size exclusion chromatography with a sample of
blood to separate hemoglobin and vitamin B12. While we can show that this is an effective
technique in separating the components in blood, the overall objective is to show how
chromatography is effective by using a Bradford assay and gel electrophoresis to indicate that
these components are in fact separated.
Background information
In size exclusion chromatography, one is given a powerful method for determining and
investigating molecular weight distribution in polymers. [1] It depends on the relative size or
volume of a macromolecule with respect to the average pore size of the packing. Usually, silicabased packings are preferred because it insures the quality of your results and high efficiency in
polymers while polystyrene gels are the most selected for organosoluble polymers. [3] These
packings have tiny holes, are porous, and are packed together inside the column. During the
process of chromatography when the buffer is added to move the proteins down the column,
the protein binds to the oppositely charged beads as these beads act as temporary traps for the
molecule of interest. Therefore, everything will pass through first before the protein we are
interested in. [1] Also, larger molecules will move through first. The beads will trap smaller
molecules, while the larger molecules pass through the column. In a mixture of vitamin B12
and hemoglobin, we can expect hemoglobin to pass through first because it is a larger
molecule.
The Bradford assay is based on the reaction of Coomassie G-250 with amino acids in
proteins to measure the amount of protein in sample [4]. When we combine the dye and
protein samples in a cuvette, there will be a color change in the dye from and reddish-brown to
a brilliant blue. This color change occurs due the protein binding changes the dye from its
cationic form to the anionic form at the 595 nm end of the spectrum [2]. Generally, the brighter
the blue color, the higher the protein content in the substance. While one may be able to guess
the absorbance value, we can see exact values when using a spectrophotometer. A standard
curve will have to be used with the values of proteins in the cuvettes to help evaluate any
unknown protein concentrations of the sample.
Gel electrophoresis would be the next step after using Bradford assay. Gel
electrophoresis has been used has a helpful tool in the birth of proteomics because of its ability
to separate and determine relative size of different proteins. SDS electrophoresis was made
popular in the 1970s by protein biochemists after a successful electrophoresis coupling
denaturing IEF to SDS PAGE, which stands for sodium dodecyl polyacrylamide gel
electrophoresis. [5] After running the gel, one can measure protein bands and see how far they
traveled along the gel from the negative to the positive end. The pore size of the gel decreased
as the gel run from the negative to the positive end, so smaller sized molecules will travel
farther. When a protein is too large to pass, it will stop, creating a line on the gel. It is important
to create a standard or ladder to compare the other proteins to so you have find molecular
mass after finding standard curve equation. The standard curve should have a negative linear
trend because the farther the protein travels, the smaller the molecular mass.
Results
Table 1: Examination of collected fraction
Tube #’s
Color
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
No color change
No color change
No color change
No color change
Strong blue
Tube 6
Tube 7
Tube 8
Tube 9
Strong blue
Light blue
Light blue
No color change
Tube 10
No color change
Table 2: Standard Curve Absorbance Values
Sample
Std. #1
Std. #2
Std. #3
Std. #4
Std. #5
Std. #6
Std. #7
Abs @595 nm
0.083
0.163
0.361
0.537
0.636
0.707
1.027
Concentration (mg/mL)
0.125
0.25
0.5
0.75
1
1.5
2
Table 3: Spectrophotometric data for collected fractions
Figure 1: Standard curve:
Tube #’s
Tube 1
Abs @ 595 nm
0.332
Tube 2
Tube 3
0.018
0.022
Tube 4
Tube 5
0.501
0.749
Tube 6
Tube 7
Tube 8
Tube 9
Tube 10
0.923
0.382
0.273
0.095
0.227
Table 3: Final Concentration of Unknown Samples
For the final concentrations, we can use the formula y=0.4711x+0.0898 to find each of
the approximate concentrations for each sample. We had to calculate these values without the
equation, so we drew a line of best fit and found approximate concentrations for the
unknowns. After finding the concentration of each, we measured up with the pipettes to
determine how many uL (microliters) were in each. To find the ug (micrograms), one can use
the equation ug=concentration x microliters.
Tube #’s
Tube 1
Abs @ 595 nm
0.332
Concentration
≈0.4
uL in each tube
210
ug in each tube
84
Tube 2
Tube 3
0.018
0.022
≈0.090
≈0.095
75
45
6.75
4.275
Tube 4
Tube 5
Tube 6
0.501
0.749
0.923
≈0.70
≈1.53
≈1.87
65
55
36
45.5
84.15
67.32
Tube 7
Tube 8
0.382
0.273
≈0.52
≈0.20
125
270
65
54
Tube 9
Tube 10
0.095
0.227
≈0.130
≈0.30
115
315
14.95
94.5
Table 4: Volume of Laemmli buffer added in each sample to make final concentration 0.5
µg/10 µl.
For this portion, one can use the formula C1V1=C2V2 to find microliters of Laemmli
buffer to add to each tube to give a dilution of 0.05 ug/uL for each tube. The values on the next
page are the amounts added when using this equation for each tube. We had to manipulate a
few of these values later and remove some of the volume from the original volumes because
the volume of Laemmli buffer was too great to fit in a centrifuge tube. The appropriate values
were calculated to what the micrograms and microliters would need to be.
Tube #’s
uL of Laemmli buffer added
Tube 1
Tube 2
Tube 3
Tube 4
Tube 5
1680
140
80
920
1683
Tube 6
Tube 7
Tube 8
Tube 9
1346
1300
1080
300
Tube 10
1890
Conclusions
These tubes were then allowed to rock and then pipetted into each of the wells on the
gels. There were 5 uL of the samples added into each well per lane. Once the gels ran and were
allowed to sit, there was no band formation. This could have occurred for a couple of different
reasons. First of all, the samples could have been too diluted. Although we calculated them
appropriately, the dilution factor may have been too low. If we increased the dilution factor,
perhaps the bands may have formed. The lack of band formation also could have been
attributed to there not being enough sample volume added in each lane. If we had increased
the volume added, we may have had band formation occur. Both of these reasons could have
contributed to the reason no bands formed. If we increased dilution factor and added more to
the wells, we may have bands form.
References
1. Boymirzaev, A.S., Turavev, A.S. (2010). Non-exclusion Effects in Aqueous Size-Exclusion
Chromatography of Polysaccharides. Chinese Medicine 1, 28-29.
2. Carlsson, N., Kitts, C.C., Akerman, B. (2012). Spectroscopic characterization of Coomassie
blue and its binding to amyloid fibrils. Anal Biochem 420, 33-40.
3. Howard, G.B., Barry, E.B., Jackson, C. (1994). Size Exclusion Chromatography. Anal Biochem
66, 595-620.
4. Ku, H.K., Lim, H.M., Oh, K.H., Yang, H.J., Jeong, J.S., and Kim, S.K. (2013). Interpretation of
protein quantificatin using the Bradford assay: comparison with two calculation models. Anal
Biochem 434, 178-180.
5. Rabilloud, T., Chevallet, M., Luche, S., Lelong, C. (2010). Two-dimensional gel
electrophoresis in proteomics: Past, present, and future. Journal of Proteomics 73, 20642077.