protein samples running in a straight line

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: honeev, Leonid, amiradm, BioTeam

Post Reply
curious1
Garter
Garter
Posts: 23
Joined: Mon May 15, 2006 4:45 pm

protein samples running in a straight line

Post by curious1 » Tue Sep 26, 2006 6:14 pm

Hi everyone,

I am recently running SDS gels with protein samples for western blots. However, I noticed that my gels have been running strangely, that is the protein samples are all running in a straight line! Has anyone ever experienced this before? What could this be from? Please advise!

User avatar
MrMistery
Inland Taipan
Inland Taipan
Posts: 6832
Joined: Thu Mar 03, 2005 10:18 pm
Location: Romania(small and unimportant country)
Contact:

Post by MrMistery » Wed Sep 27, 2006 6:36 pm

hmm, maybe a problem with how you made your gel... Maybe there is too little poliacrylamide-bisacrylamide, so every protein simply passes through...
Almost impossible to tell, since a milion things might cause that probably...
"As a biologist, I firmly believe that when you're dead, you're dead. Except for what you live behind in history. That's the only afterlife" - J. Craig Venter

User avatar
chemistry_freako
Coral
Coral
Posts: 193
Joined: Wed Jun 08, 2005 3:31 pm
Location: Somewhere between those molecules
Contact:

Post by chemistry_freako » Thu Sep 28, 2006 2:38 am

Have you tried looking through your protocol and analysing what are the possible things that could have gone wrong? :wink:
化学は、最高です

Image

curious1
Garter
Garter
Posts: 23
Joined: Mon May 15, 2006 4:45 pm

Post by curious1 » Thu Sep 28, 2006 5:13 pm

Hi,

I used a 15% gel so the acrylamide concentration should be sufficient, this is the first time I'm doing a western blot and no matter how many times I read the protocol I just seem to have a million questions and uncertainties. Seems like nothing is going the way it's supposed to. So I stained the straight line gel to see how it would affect the proteins and it turns out I could see the proteins just fine. So I went ahead and ran another gel, this time same thing with the straight line but since the gel turns out fine as I had checked I just moved on. I did the overnight transfer and stained my gel to make sure there was a complete transfer, but I see some protein still left on my gel. However, I see that the ladder had gotten transferred over to the membrane blot so hopefully some protein got transferred at least. I am also curious to know whether people have used two different antibodies on the same blot. That is, if I want to use actin as a control can I first incubate with actin antibodies and then expose the blot and then re-blot with my target antibodies? Some people say I can just wash off the blot and re-probe if the two different antibodies are different enough in size (mine is 18kd and the actin is about 50kd), some say you need to "strip" the blot of the first antibody, and still others say that I can actually dump both antibodies in at the same time during incubation. Who can I trust? Sorry if I sound frantic because in fact I am!

Post Reply

Who is online

Users browsing this forum: No registered users and 4 guests