Is My Protein Pure or Not? - Protein purification, SDS-Page (Feb/11/2011 )

Try to talk to your colleague about his/her application. If your colleague do not require a native protein, you can just use your current column purified products and do SDS-PAGE, followed by excise the band and do electro elution. After the electro elution, you can concentrate the protein by using vivaspin protein concentrator (column with membrane). Do a protein quantification (BCA, Bradford etc...), and dilute to the concentration you want.

LOLx, I'm also a dummies as well...

rpjkmust916 on Thu Feb 17 02:22:57 2011 said:

the scanned image of the gel can be brought into a program like imagej and the lanes can be evaluated like they were scanned. each band can be integrated and given a value relative to the amount of stain incorporated (you can, if you know how much protein was loaded, determine the actual mass for each band). you can then determine the percent purity based on the whole lane and the ratio of the two bands of interest.

the ratio would be important to determine if the two bands are subunits of the same protein, if they maintain the same ratio for all samples then they may be. from what you say in the post about the formula weight of your protein, they are probably not subunits.

you can determine if the lower band is a fragment of the upper band by western blot if the remaining protein contains the epitope(s) recognized by the antibody.

mdfenko on Thu Feb 17 21:39:24 2011 said:

Thanks mdfenko,
I will learn how to use the ImageJ


Adrian,

Thank you for ur advice....

Welcome, hope this helps.

some might be satisfied with CCB-stained gels to state purity however staining with silver or nanogramm-sensitive dyes is the correct method to state purity on the gel level...

rpjkmust916 on Fri Feb 11 08:46:25 2011 said:

Inmost sun on Sat Feb 19 15:03:14 2011 said:

Thanks Inmost sun for your suggestion...unfortunately in my lab only Comassie blue stain available....

May I ask about the electroelution?
I've read several papers about electroelution...looks like it require some specific apparatus. Is there any simple method that I can use to run electroelution?
Any suggestions, please?

rpjkmust916 on Tue Feb 22 12:24:07 2011 said:

while silver stain is very sensitive and qualitative, it is too variable between different proteins to use for quantitation (and, therefore, percent purity). coomassie brilliant blue r-250 (not as variable as g-250) is well suited for this purpose. you may need to load more to see minor contaminants but they are minor and will not significantly influence percent purity determinations

all you need is dialysis tubing, horizontal gel apparatus and a suitable buffer

Thanks mdfenko,

Can I use the running buffer that I've used to purify the protein with IMAC column to do the electroelution?
Or do I have to find a different type of buffer?

May I know, how much the concentration of target protein that you've usually recover from electroelution?

Thank you very much for spending time in answering my questions.

rpjkmust916 on Wed Feb 23 01:34:02 2011 said:

standard electrophoresis buffers work well, others you may have to try yourself

concentration depends on how much protein is in the band being eluted and the volume of buffer in the dialysis bag. you can recover most of the protein from the gel.

Thanks mdfenko