Hello guys,
I hope that someone could help us with this.
In our lab we have a purified fungal xilanase from Colletotrichum graminicola that has 20 kDa of estimated mass. The total content of carbohydrates was estimated in 97% with Dubois method. Several samples was applied on SDS-PAGE 10% and we have found deformed bands when the samples was concentrated before gel application (see attachement). The protein was diluted in salt-free water and concentrated 160-fold (30 ug total) before gel application. Anyone has seen this pattern of glycoproteins migration in SDS-PAGE gels? Any suggestions?
In the attchment we see on the second and fouth lanes the band of the deformed protein and in the third lane we see the molecular marker standards
Yes, I have seen band morphology like this before but it was further down the lane, funneling to a point at the end of the gel, and much more of the band was stained; it was an accidental result of over-loading the lane. Did you actually load ~30 ug into the lane? What was used for staining? Sometimes an overloaded lane doesn't stain well with Coomassie and extensive post-translational modifications will also have an impact
Notice that the MW std lane is also distorted as though something from the flanking lanes is moving into the MW lane during the electrophoresis. Also notice that contaminants from your preparation seem to have spilled over into both the MW lane and lane 5 as though the entire well of lane 4 was filled with sample. I assume you used a conventional Laemmli heat-procedure for preparing your sample?
The way you describe the glycosylation, are you writing that 97% of the protein's mass is glycosylated (the math won't work) or that 97% of the enzyme molecules are glycosylated and probably to varying degrees? High glycosylation or PEGylation can affect band morphology where the typical shape is inverted and with fuzzy edges due to the varying Stoke's radius from the carbohydrate/PEG. For a fungal enzyme purification you probably followed a standard/simple purification scheme without using less common buffer components, so what was the reason for the dilution and relatively high fold concentration: high salt or ethylene glycol from a HIC step? So what was the last purification step and what buffers were used?
It looks like there was a lot of something other than your protein in the sample. This may have arisen during the concentration if something was concentrated besides the protein. This can happen with detergents because the micelles of some detergents do not pass through the ultrafilter. PEG is another possibility.
Yes, I have seen band morphology like this before but it was further down the lane, funneling to a point at the end of the gel, and much more of the band was stained; it was an accidental result of over-loading the lane. Did you actually load ~30 ug into the lane? What was used for staining? Sometimes an overloaded lane doesn't stain well with Coomassie and extensive post-translational modifications will also have an impact
Notice that the MW std lane is also distorted as though something from the flanking lanes is moving into the MW lane during the electrophoresis. Also notice that contaminants from your preparation seem to have spilled over into both the MW lane and lane 5 as though the entire well of lane 4 was filled with sample. I assume you used a conventional Laemmli heat-procedure for preparing your sample?
The way you describe the glycosylation, are you writing that 97% of the protein's mass is glycosylated (the math won't work) or that 97% of the enzyme molecules are glycosylated and probably to varying degrees? High glycosylation or PEGylation can affect band morphology where the typical shape is inverted and with fuzzy edges due to the varying Stoke's radius from the carbohydrate/PEG. For a fungal enzyme purification you probably followed a standard/simple purification scheme without using less common buffer components, so what was the reason for the dilution and relatively high fold concentration: high salt or ethylene glycol from a HIC step? So what was the last purification step and what buffers were used?
Check the level of upper chamber buffer, there might be leaks during electrophoresis, I usually fill it with Milli Q H2O and leave on the bench for about 5 min to check for leaks.
Good luck
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