I fused the GFP to my protein of interest. I know their MW separately, but I would like to know the MW of my protein with GFP. I know there are some websites to calculate the MW of proteins using aa sequences, but I am not sure how I can get MW of two fused protein from these websites. Does anyone have any idea? Thanks.
Thanks
Dear Ozkan, As you mentioned you can use a lot of free online calculators for determination of proteins MW.
e.g.
http://pir.georgetown.edu/pirwww/search/comp_mw.shtml
http://protcalc.sourceforge.net/
http://web.expasy.org/compute_pi/
http://www.bioinformatics.org/sms/prot_mw.html
But notice that, some need protein AA sequence and some need importing data from UniProt Knowledgebase (Swiss-Prot or TrEMBL),...
Finally it may be post traslational modifications changed the MW.
So it shows only stimate of MW related to fused proteins.
And you can check it experimentally by Gel filtration, or electrophoresis too.
best
mehdi
Dear Ozkan,
I agree with mehid suggestion it is better to form wet lab experiment because exact PTM's can not be calculated with tools. simply pull down your protein by following antibodies either GFP's or your protein specific then run on gel and result is in your hand and mehid aslo suggested for chromatography so you can also go with that.
Because calculation by tools is comes by simple formula and they do not think about PTM's.
try go with wet lab experiment.
Best Wishes
Hi Ozkan,
Is there any way to construct a fused protein using in silico tools.
daltons are a measure of mass. If you fuse a 10kg weight with a 5kg weight, what is the new weight? It is, of course, 15kg. protein folding has no effect on its mass, just as 1kg of lead weighs the same as 1kg of feathers.
Some of the other answers are talking about the observed mW by SDS-PAGE, which may differ slightly.
I agree with a calulated measurement since, as pointed out above, wet experiments such as SDS-PAGE are subject to artifact from incomplete protein denaturation. A calculated MW is only accurate if you know the fusion sites. If I created the fusion protein myself, I usually take the coding region and convert that via online tools into the a.a. sequence, then use that to calculate the fusion protein MW. There are some wet experiments that are more biophysical such as mass spectrometry and a.a. sequencing, but they require protein purification and cost of analysis.
Hi just to add a very "crude" way of calculating the weight of a protein is to assume an "average" of 110 Daltons per residue. So if the primary amino acid sequence is 100 amino acids this will be ~ 11 kDa. (GFP = 238 amino acids which cones out as: 26.1 kDa which is pretty close).
Obviously if you have access to the web you can calculate the mass exactly using one of the free calculators listed above. It's always worth bearing in mind that the mass given will be for the unmodified protein - glycosylation etc can alter the weight and also cause it to run differently on a gel.
Hope this helps,
Gary
http://web.expasy.org/compute_pi/
http://www.expasy.org/proteomics
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