Gel filtration chromatography or perhaps just gel filtration is used to separate or purify protein based on the size properties. These protein include enzyme, polysaccharides, nucleic acids and other biomolecules. There are two kinds which technique can be applied. One is for group separation, and second is for high resolution fractionation of biomolecules.
Group separation basically employs the gel matrix size range to remove or to obtain specific molecular weight size of different proteins. On the other hand, high resolution fractionation is used to isolate one or more components and also to determine molecular weight distribution. I myself had done the latter.
Gel filtration is a robust technique and is able to handle chemicals sensitive to changes in pH, concentration of metal ions or other harsh environmental condition.
So these are my steps when I conducted my GFC. Simple introduction is given at previous post, gel filtration chormatography. I put the link of complete GFC procedure on that post. It must be noted that what I put here is based on my own experience to determine molecular weight distribution of humic acid (organic matter) not purifying protein. Each of them have each treatment.
I used a C 26/70 Amersham Column, it is an empty column so it is much cheaper besides I already have the gel Sephadex G-75TM. So again make sure you know what column you need. I suggest you purchase a prepacked column, it will save much time in your side, since you do not need to set up from the scratch like me. For me, it takes almost 2 days just to set up until I equillibrate the column.
First, prepare the column and clean them with buffer. There are two buffer usually used, Tris Buffer and Phosphate Buffer Solution (PBS). I am using Na2H2PO4 pH 7 with NaCl 0.1 M. So clean the column with the buffer, inside and outside, if neccessary sink the column into the buffer. But I did not do that, simply because the column is big.
Second, install the column vertically with column handler. Don’t forget to place outlet connecting tubes. I did not use inlet tubes, since I was relying on gravity pressure. So I opened the inlet top end piece. Always refer to your column booklet if you find unfamiliar term in my post.
Third, prepare the gel Sephadex G-75. I prepared around 400 mL of buffer in a beker glass, I poured the gel powder carefully until it filled around 300 mL, thats almost 75%. I let the powder swelled for about 3 hours under room temperature.
Fourth, I prepare solution standard of Polyethylene Glicol (PEG) with different sizes 72,000; 35,000; 15,000; 6,000 and 1,500 Dalton with of course Blue Dextran 2000. Each of them is 3 mg/mL, as for Blue Dextran was 0,2 mg/mL. I prepared approximately 50 mL of solution standard, although I only need 3 mL. But thats okay, who knows I will need to repeat to standard calibration.
Fifth, after 3 hours and your gel is ready, I pour buffer until it gave 2 cm above the column bottom base. I gently swirl the gel, I pour the gel solution slowly to the empty column. You will notice that the gel is slowly moving down when it form packed bed. You probably have to wait 50 or 60 minutes. I measured the bed column which was around 300 mL, then I equilibrated the column with around 400 mL buffer under 2 mL/min flow. I suggest you equilibrate with 2-3 times bed volume.
Sixth, before I inject the standard solution, I pipetted first the buffer above gel surface, leaving only 0.5 cm of buffer above bed gel. I inject 3 mL solution standard, I run the buffer. As soon as the standard (marked with blue dextran) completely entered the gel, I start collecting 6 mL sample under 2 mL/min flow until 300 mL sample is collected. If your gel volume is 300 mL then your total sample would be 300 mL as well.
Seventh, I measure each of the fractions with UV254. Put in excel worksheet these rows, elution volume (every 6 mL) and UV254. Identify the peaks, the first peaks will of course be Dextran, 2,000,000 Dalton. Followed by 72,000; 35,000; 15,000; 6,000 and 1,500 Daltons. Plot in logarithmic scale, y is log molecular weight, x is elution volume. You have the formula now to determine the molecular weight.
Eighth, Inject the sample, do the same as above. Plug the elution volume to the formula from number seven above, and voila! you have the sample molecular weight.
Thats it, I know this is not perfect, I even think you are still confused. If you have something in mind, let me know. Below is the source I got for this experiment.
http://faculty.samford.edu/~gekeller/kennedy.html
http://en.wikibooks.org/wiki/Proteomics/Protein_Separations_-
_Chromatography/Molecular_Exclusion
http://newarkbioweb.rutgers.edu/bio301s/
Lab%202-%20pH%20-buffers.htm
http://newarkbioweb.rutgers.edu/bio301s/
Lab%204-%20mol%20wt-%20column%20chromatography.htm
http://www5.gelifesciences.com/aptrix/upp01077.nsf/Content/
ProductsTree
http://www.biology.ualberta.ca/facilities/multimedia/uploads/
procedures/video/sephadex.html
I have to prepare a 30 cm column using sephadex g-25. I cannot find an empty column to do this. can you help. I do not need to know the mol wt but just to purify my product from small mol wt impurities. I presently use a 13 cm column which gives me separation but the volume that I can apply is limited. can you suggest something?
You can try getting a XK 16/40 or XK 26/40 from GE healthcare, ths columns come with an adapter so you can pack columns of 40 cm and below.
Hi, I want a use a sephacryl S300 to aislate diferent fracction of hidatid liquid. I want to kwon how calibrate the column and how obtain the Chromatogram.
Thank.
JoLu