I have some polysaccharide fractions obtained by DEAE-sepharose column separation. I wish to determine its approximate molecular weight by caring out SDSPAGE. However I cannot obtain nice bands. Please help me if anyone knows how to do this.
Given below is the procedure that I used
Markers.
dextran sulfate, 500 kDa
chondroitin 6-sulfate, 60 kDa
chondroitin sulfate, 50 kDa
dextran sulfate, 8 kDa
Given bellow are the conditions that I used to prepare the gel. However I carried out the electrophoresis using the same running buffer that they use for proteins.
Separating gel
DW 5.3 ml
30% acrylamide mix 2 ml
Barbital buffer 0.02 mM (pH 8.6) 2.5 ml
10% SDS 0.1 ml
10% ammonium persulfate 0.1 ml
TEMED 0.008 ml
Stacking gel
DW 3.442 ml
30% acrylamide mix 0.833 ml
Barbital buffer 0.02 mM (pH 8.6) 0.625 ml
10% SDS 0.05 ml
10% ammonium persulfate 0.05 ml
TEMED 0.005 ml
Electrophoresis – 30 min at 100v
Staining
0.1% toluidine blue in 1% acetic acid – keep for 30 min (with continuous shaking)
De-staining
Wash for 10 min 20 min 30 min 4h in 1% acetic acid (with continuous shaking)
Thank you
Hi,
I have no experience in the SDS-PAGE separation of polysaccharide, but in proteins it is difficult to have good separation of such wide range, from 500 to 8 kDa. On the other hand, 6% separating gel seems to be low, and you did not indicate the acrylamide/bisacrylamide ratio of your mix
Have you try a gel around 7.5%? what is the ratio of your acrylamide/bisacrylamide mix?
Are 30 min enough?
What is the appearance of the bands that you obtained in the marker lane and in the samples?.
You said that they are no nice, but do you obtain any band?
Are you sure that the polysaccharides are eluted from the DEAE-sepharose?
Good luck!
Polysaccharides generally tend to have a range of molecular weights, rather than a specific MW like proteins or even oligosaccharides. I'm afraid that no matter how good your PAGE may be, you will always get broad bands or smears. Polysaccharide MW measurements are usually done by HPSEC or light scattering, or a combination of the two. More recently, FFF (Field flow fractionation) has been used successfully. Even the standards you mention do not have specific molecular weights, but MW ranges instead. Similarly, electrophoresis depends on the charge density of the molecules. As they naturally occur, many sulfated polysaccharides do not have exactly the same number of sulfate groups, so you will again have a range of mobilities that depend on both the variations in MW and the variations in charge density. I'm not even sure that SDS will interact with sulfated glycans in the same manner they interact with proteins. All things considered, I would recommend using an alternate method.
Greg is absolutely correct, polysaccharides are generally very poydisperse so you will not get tight bands on a gel like you do with proteins. Oligosaccharides (in particular N.glycans) have been analysed by gel electrophoresis (this is typically called FACE: Fluorophore Assisted Carbohydrate Electrophoresis). Originally there were commercial kits available for that. More recently FACE has moved to capillary electrophoresis, but for high mw polysaccharide I thinks you will always have the polydispersity issue.
I will agree with Dr. Greg cote
Dynamic light scattering studies should be preferred for MW of polysaccharides
I agree with Dr. Cote, both plant and microbial polysaccharides are very disperse, so we usually speak of "number average" or "weight average" molecular weights. Methods like Multi-Angle Laser Light Scattering - Size Exclusion Chromatography give very good results for the determination of molecular weight distributions.
Thank all of you for your nice comments and advice. I also had some doubts about the validity of this method. But I gave it a try.
I tried it for 2 times changing the concentration of toluidine blue since the first time it took almost 5 days to de-stain. But both time I got 2 bands for 8 kDa and 60 kDa one and the 8 kDa one was strangely dispersed (lengthened) horizontally and both were not much clear.
Anyways now that I'm confidant with the reasoning and the errors with this method I would try to send the samples for a suitable analysis. Again thank you all so much for the v. nice advice.
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