best and simple method to purify protein - (Aug/03/2010 )

mdfenko on Tue Sep 28 20:15:28 2010 said:

Tq for helping me during my project....
can you explain why my gel look like the attached pic??

sample load may not be sufficient to obtain strong banding. background "smearing" may be due to incomplete denaturation (it may also be the reason that the bands are weak.

have you tried silver staining?

mdfenko on Fri Oct 1 18:04:54 2010 said:

hi...i didn't used silver staining b'coz lack of reagent....i'm just used commasive blue stain....
in my discussion i should write about i can't get a good band because of incomplete denaturation???
using attached pic can i estimate my protein kDA?

TQ

intan on Fri Oct 8 07:32:01 2010 said:

i'm not sure you need to mention the background. just the analysis of the bands (which should be useful in determining an approximation of the molecular weight).

how can i determine what type of protein in my sample???based on their molecular weight..

Molecular weight is not specific enough for precise identification. It would work only if you already have some paper or database information on protein pattern of of your certain species of fungi.

To identify protein you need to isolate it and get sequence of N-terminus or mass spectrometry analysis.

i'm doing protein profile of mycelia n fruiting bodies of mushroom..now at last stage...after sds page and calculate mw of protein based on protein ladder, my lecture suggest to check what type of protein in my sample based on mw...but i don't know how to find it because i don't have protein sequences..in your opinion what should i write in my thesis for result n discussion?


TQ

If your work don't include sequencing or mass spectrometry, whatever you do will be very general but if your adviser says it's ok - do it.

You may start with Uniprot search for your species of fungi.

i got it !!!
thanks a lot for helping me...

may God bless....