Omnisec Protein Q&A

Recently, I’ve presented a new webinar focusing on the protein characterization using the OMNISEC, the highest quality Gel Permeation Chromatography (GPC) / Size Exclusion Chromatography (SEC) system for the measurement of absolute molecular weight, molecular size, intrinsic viscosity, branching and other parameters. Thank you to all participants for your interest and attention during the webinar.

In this webinar, I highlighted the key features and benefits of the OMNISEC system. How to run samples and analyze data and how to access repeatability of multiple sample measurements.

As usual, after the live section of the webinar, I field the questions from the audience but as I couldn’t answer them all, I compiled the list for you.

Did you use a sub 2 micron column for the IgG separation?­

The column used in these measurements was s single 300 A column with a particle size of 3 um.  It was an analytical column 300 mm long x 15 mm ID.

Do you have an example of an IgG with a post peak shoulder as a minor species that you have been able to determine the M.W of?

­I’ll assume here that you are asking about detecting fragments in the presence of the IgG.  This is certainly possible but more dependent on the column resolution than anything else.  I’ve certainly studied IgG fragments but don’t have an example exactly as you described that I could show.  If you are interested in such samples, though, I’d be happy to measure some and see what information we could get.

­What about the recoveries? for the heated IgG they were 50% what does this mean? I don’t remember the recovery of the IgG but I would like to know what low recovery means and how it can be solved. ­

The recoveries for both of the IgG samples (native and heated) was just over 50%.  The most likely explanation for this was simply that I dissolved the lyophilized powder for a short period and that it was not fully dissolved and some sample was subsequently lost, either when I filtered it before injection, or on the column.  Better practice would have been to use a UV spec. to measure the sample concentration before injection.  Then I would have a better idea if any was lost on the column.  In that case, either a different column or mobile phase would improve the sample recovery.

­When selecting peaks, does the software automatically determine the peak limits (peak width) for integration or does it fully depend on the operator?­

The software integrates the full area of the selected peaks so the results cover that full range.The only limitation here is when one of the detector signals returns to baseline before the edge of the peak.  In this case, the software will extrapolate the molecular weight trend in that region.  The main body of the peak is using the real calculated data though.

What column did you use­?

These measurements were performed on a 300 A silica column with a 3 um particle size.  We supply these columns and call them PLS3030.

My protein is 12 kDa large, what will be the minimal protein Load? 

Of course, a lower molecular weight protein like this will scatter less light so we might need a bit more sample but we can see the signals here are very strong.  From other measurements we’ve made with peptides, I think we could make a measurement with somewhere around 5 µg of sample, possibly a little bit less.

Please do not hesitate to contact me if you have any further questions that haven’t been answered above!

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