Tuesday, April 3, 2012

What I've been doing lately...

Isolation and Characterization of Bovine Milk α-lactalbumin 

 February 28, 2012 
(and seriously, you steal this... you DIE. *ahem* it's called plagiarism and you don't want to know how many hours I spent working on this thing. I care if you steal it.)

 Abstract
 The ability to isolate and characterize alpha-lactalbumin has potentially important links to discovering the cure for things such as Type II Diabetes and Autism. Here, whey was purified from milk, affinity and size exclusion chromatography were used to separate out alpha-lactalbumin and beta-lactoglobulin, and an SDS-PAGE and a Bradford assay were performed. Molecular weight of alpha-lactalbumin was found to be between 9,436.37 D and 15,131.4 D, and compared to the known weight of 14,200 D; molecular weight of beta-lactoglobulin was found to be 17,317 D and compared to the known weight of 18,400 D.

Introduction
 Bovine milk, a substance providing necessary nutrients for human development, can be divided into whey and caseins. Caseins, which appear in cheese and paint, are also of interest in autism studies, especially in how a casein-free diet affects autism. Whey, the other main, though lesser, component of milk, stimulates insulin release and is involved in regulation of blood sugar.

Whey is made up of about 58% beta-lactoglobulin, 13% alpha-lactalbumin, and 12% other proteins. The ability to purify proteins can be of utmost importance in studying things such as diabetes and autism. In this case, various methods were used to purify and characterize the proteins found in milk in order to estimate the molecular weights of alpha-lactalbumin and other proteins. By bringing a protein to its isoelectric point, it will precipitate out and can then be separated. Centrifugation, preceded by acid and heat-induced precipitation, can be used to remove caseins from milk, leaving behind the whey, which can then be further separated into alpha-lactalbumin and beta-lactoglobulin.

Affinity and size exclusion chromatography are used to separate them further. Size exclusion chromatography – using Sephadex gel filtration methods – allows the beta-lactoglobulin to run through faster, while the alpha-lactalbumin is caught along the way. Affinity chromatography – using a Cu(II)-IDA-agarose affinity column – is more specific, targeting one type of protein. Since alpha-lactalbumin is a metalloprotein, it binds to metals and can then be eluted with something that will break the copper-alpha-lactalbumin bond . Following purification, UV spectroscopy and SDS-polyacrylaide gel electrophoresis can then be used to further characterize the protein. Performing SDS-PAGE separates all proteins by molecular weight only, with smaller molecules traveling further down the gel. A Bradford protein assay gives the absorbance and can be used to find the protein concentration . 

Materials and Methods 
 One hundred mL of nonfat milk was centrifuged at 16,00g for 45 minutes in a refrigerated centrifuge, after which pH of the supernatant was adjusted to 4.5 with dropwise addition of .5 M HCl. The solution was then heated at 37oC for 30 minutes while being stirred, before being centrifuged at 16,000g for 30 minutes. A .45 µm syringe cartridge filter was then used to clarify the supernatant, resulting in isolated proteins.

 Chromatography was then performed, using Sephadex G-25 fine mesh and a 100 mL slurry of .02 M Tris at a pH of 7.0. Baseline was established and then four mL of whey was added. More buffer was run through column using an eluting solvent supply that kept a constant solvent flow into and out of column. There were two peaks and it was run back to baseline. A fraction collector was used to collect one mL fractions at a flow rate of 10 mL/hr. Absorbance of selected fractions at 280 nm was then measured.

 Affinity chromatography was performed at the same time, using an IDA-agarose prepacked column. Ten mL of buffer A, composed of .02 M Tris and .05M NaCl at a pH of 7.0 was used to wash column before .5 mL of .1 M CoSu4 solution was added and allowed to enter gel. Buffer A was again used to wash column until all excess Cu(II) had eluted, and then drained just to the top of the gel. A .5 mL portion of whey was then added and 1 mL fractions were collected in individual test tubes. One mL more of buffer A was added once whey entered gel, and then column was filled with Buffer A. Once this eluted out, 10 mL of buffer B, composed of .02 M Tris, .5 M NaCl, and .02 M imidazole, at a pH of 7.9, was added to the column to elute α-Lactalbumin. Absorbance of all fractions at 280 nm was then measured.

 The top two fractions from each peak found after both chromatographies were finished were then used for a Bradford protein assay. Using 10 µm of fractions A, B, E, F, G, H, 20 µm of fractions C and D, 790 µm of water for all fractions but C and D, which used 780 µm, and 200 µm of Dye for all fractions, the absorbency was used to find the concentrations of all unknowns. A standard curve was made using five standard concentrations from 1.6-25 µg/mL.

 An SDS-PAGE was also performed using .6 to 11 µg of protein, with 4% acrylamide stacking gel and 12% acrylamide resolving gel, run for 70 minutes at 150v. The slab was then soaked in dye soluion of .25% Coomassie Blue in methanol-acetic acid-water (5:1:5) for 30 minutes before gel was removed and an acetic acid-methaol-water (7:7:86) solution was used to destain the gel overnight.

 Results/Discussion 

As can be seen in Table 1, size exclusion chromatography was used to separate the alpha-lactalbumin and beta-lactoglobulin and the absorbency of the peaks found.


Table 1: Absorbency of Fractions from Sephadex 
Chromatography at 280 nm
Fraction # 7 8 32 33
Absorbency   2.18 2.20 1.94 1.99


This information was used to create a graph (Figure 1) of the absorbency of the fractions over time, showing a peak for both alpha-lactalbumin and beta-lactoglobulin.

 Absorbency of Sephadex Chromatography Fractions at 280 nm vs Time 

 Figure 1: Purification of alpha-lactalbumin and beta-lactoglobulin by size exclusion chromatography through Sephadex G-50 fine mesh pre-equilibrated with a 100 mL slurry of .02 M Tris at a pH of 7.0. 

The proteins were eluted at a flow rate of 10 mL/hr and fractions of 1 mL each were collected and assayed. Another graph (Figure 2) was created using data from the more specific affinity chromatography. Again, two peaks can be seen, one from alpha- lactalbumin and one from beta-lactoglobulin.


 Figure 2: Purification of alpha-lactalbumin and beta-lactoglobulin by affinity chromatography using an IDA-agarose prepacked column. >5 mL of .1 M CoSu4 solution, .5 mL of whey, a buffer composed of .02 M Tris and .05M NaCl at a pH of 7.0, and a second buffer composed of .02 M Tris, .5 M NaCl, and .02 M imidazole, at a pH of 7.9, were added to the column to elute α-lactalbumin. One mL fractions were collected and absorbance of all fractions at 280 nm was measured. 

Results of the SDS-PAGE can be seen in Figure 3.

 Figure 3: Results from SDS-PAGE, with 4% acrylamide stacking gel and 12% acrylamide resolving gel, run for 70 minutes at 150v. Ladder shows Myosin, Beta-galactosidase, Carbonic Anhydrase, Soybean trypsin inhibitor, Lysosyme, and Aprotinin; BSA did not appear. Values from the ladder appeared as they should, with the exception of BSA, which was not visible. 

As expected, many proteins showed up in the lane for whey. For lanes A and B, size exclusion lanes, larger molecular weights were expected to appear – and the wide band in lane A appears to be alpha-lactalbumin. Band in lane B, however, appears much lower and more faintly. This could be due to errors in loading the gel or other errors associated with protein concentration. Smaller molecular weight proteins were expected in lanes C and D, and in those columns no large molecular weight proteins appear. Any other proteins should show up in lanes E and F, while lanes G and H, from affinity chromatography, should show alpha-lactalbumin. Lanes E and F do show various other proteins, and as expected, lane G showed a thicker mark for alpha-lactalbumin, as well as a few other proteins still washing out, while in lane H a faint mark from alpha-lactalbumin can perhaps be seen. Lack of visibility is probably due to a low concentration of proteins. The ladder MW was then used to construct a standard curve in order to find the MW of the unknowns. However, since not all bands showed up, the ladder may be prone to error.

 Figure 4: SDS-PAGE Standard Curve from Rf values and the log of the molecular weights from ladder; also showing equation used to find molecular weights of other proteins.

Using the SDS-PAGE Standard Curve, molecular weights were then found for each band of proteins showing in the SDS-PAGE. From Table 3, molecular weight of alpha-lactalbumin – appearing as a the wider bands in Figure 3 – appears to be 13,221.7 D in the lane for whey, 14,144.4 D in lane A, 15,131.4 in lane G, and 9,436.37 in lane H. Given the error associated with lane H, it makes sense that that number is the furthest off from the known molecular weight of 14,200 D for alpha-lactalbumin. In both lanes C and D, a molecular weight of 17,317 D is found, which is closest to the known molecular weigh of 18,400 D for beta-lactoglobulin, though still rather far off. This may be due to low concentrations of protein making reading of SDS-PAGE difficult or other errors in loading lanes.



Table 3: Molecular weights of proteins from peak absorbencies of Sephadex and Affinity chromatography, found using SDS-PAGE standard curve.
W A                B       C       D     E              F                      G        H
MW 160417 160417 160417           160417          160417
MW         140171 149952 149952               149952          140171
MW 122480 81712.2 131027           44526.7          114491
MW 13221.7 14144.4 12359.2 19818.2 19818.2          44526.7
MW 10094.9 10094.9 10094.9 10094.9 10094.9 10094.9          10094.9  10094.9
MW          8245.42 8820.82  17317 17317 21201.2 14144.4         15131.4
MW 6735.78 6735.78    10799.4 10799.4   10799.4 6735.78 8820.82 9436.37
MW 5142.08 5500.91         5142.08 5884.79



 A Bradford assay was then performed, and the concentration and the absorbance found using that concentration can be seen in Table 4.


Table 4: Absorbance and Concentration in Bradford Assay

A595
Conc. (µg/mL)
Whey
1.08
6501.12
A
.926901
1135.3632
B
.7566
590.4
C
.59557
37.552
D
.61889
74.864
E
.5871
48
F
.6549
264.96
G
.682186
352.2752
H
.688108
371.2256




 These values were then used to construct a standard calibration curve for the Bradford assay, which gave the total protein concentration. The R2 value was close to 1.0, and comparison with known standard calibration curve for the Bradford assay shows a similar figure.

 Standard Calibration Curve for Bradford Assay 

 Figure 5: Standard calibration curve for Bradford Assay from A595 and the log of the protein concentration at each assay. 

 Obvious errors include the failure of BSA to appear in the ladder of the SDS-PAGE, the extreme faintness of any protein visibility in lane H, wide variation in molecular weights found for alpha-lactalbumin – much of which could be due simply to low protein concentration. Were this experiment to be repeated, that would have to be corrected and care taken to construct a ladder with less error, leading to more accurate molecular weights. Conclusion Various methods were used to purify and characterize the proteins found in milk in order to estimate the molecular weights of alpha-lactalbumin – found to be between 9,436.37 D and 15,131.4 D – and beta-lactoglobulin – found to be 17,317 D. This was off from the known values of 14,200 D and 18,400 D. More accurate numbers could be obtained by repeating the experiment and correcting errors found, such as the lack of protein visible in lane H, and making sure no others were made. The molecular weights of the other proteins could also be used to identify those proteins and further classifications made.

 References

[1] Elder J, Shankar M, Shuster J, Theriaque D, Burns S, and Sherrill L (2006) The gluten-free, casein-free diet in autism: results of a preliminary double blind clinical trial. Journal of Autism and Developmental Disorders 36:3

[2] Boyer Rodney (2000) Modern experimental biochemistry, third edition by Rodney F Boyer. pp 59-65, 227-242 Benjamin-Cummings Publishing, Redwood City, CA.



3 thoughts shared:

Jessica said...

Please tell me I am not supposed to actually understand all of this!!!! Because I DON'T!!!! *giggle* GOD BLESS YOU AND YOUR STUDIES!!!!

Vicki said...

Good for you, girl! I wasn't familiar with the statistical methods you used in your analysis, but I got the gist of what you were doing and it looks really interesting!!

So, are you interested in pursuing research in this area once you're a doctor? I know this was just a project, but there's certainly a need for biology/chemistry research, especially relating to diabetes and autism. You'd be good at it if you did! :-)

Keep up the good work!!

Love,
Vicki

Katherine Sophia said...

Thank you, Jessica! :D It took me some time with my TA before I knew at all what I was doing... And I'm sure just flat out reading it has got to look horrible, LOL. :)

Noooo, Vicki, I really cannot stand labs or this type of experimenting. *headdesk* But at the same time, yes, I am very interested in research regarding both diabetes and autism, since they have really affected my family. I guess I'd be more interested in research that involved the actual people side of things... and also, if it was research more closely relating to either of those, I'd probably find it more enjoyable. :) But I am definitely thankful for the chance to understand the basics and see how this stuff works... I'm sure it will be useful in some way. :D

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