My protein is just 13.6 kDa so I want to see that band. who else can help me?
Thanks in advance
I've used a 10% gel to separate a whole cell lysate and obtained distinct bands ranging from 9 kDa in size to ca. 45 kDa. This is a good recipe and provides an indication of what gel % you should prepare according to the protein size you are separating: http://www.proteinchemist.com/tutorial/sdspage.html
Dear Dieter Galea
Thank you so much
i have tried to use 15% gel but my target band always run out the gel, so i thought i need more concentration of gel. Anyway, i will try to use 10% as you suggested to me. But one thing Dieter Galea, i read your document u sent to me, but they said 10%gel , we just can see the range from 20kDa-300kDa. my protein size is 13.6kDa .:(
Are you separating the 13.6 kDa protein from a mixture of proteins? Are you running a ladder? The easiest way to control distance of movement is through the run time. How long are you running the gel for?
i have not separated protein yet, and i dont know how to do that :( (sorry )
and i used protein marker ( dual color) and i run for 2hrs with 110V
Is the marker pre-stained? If so, you should get a good idea when to stop. With a 10% gel, 80 minutes work fine for me at 30 mA per gel.
yes, marker is pre-stained. i used 15% gel and it took almost 2hrs.
Do you have any idea about running buffer? low molecular weight(MW) Protein should use different with highr MW?
You can try to increase the voltage to reduce the run time (depending on what voltage you are currently usage; don't over do it) or dilute the buffer. A too concentrated buffer can increase the run time.
SDS-PAGE up to 18-20 % should do the job to see a 13,6 kDa protein, but you should use Tris-Tricine gels rather than Tris-Glycine (it is far better to look at small sized proteins).
used 1X MES buffer and run with 110 Voltage. i could see the marker from 7kDa to 26kDa, and 1 band around 16kDa but size protein of mine is 13.6kDa . Is possible that band is my target?
i ligated gene in pET28a(+) .. May be possible right? i hope that :)
Its simple. Find it here:
http://www.cytographica.com/lab/acryl2.html
or
http://www.changbioscience.com/calculator/sdspc.htm
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