protein samples running in a straight line - (Sep/26/2006 )
Hi everyone,
I'm running an SDS gels with proteins samples for western blot and the I noticed the gel is running very strangely. The protein samples have all run together in a straight line! Has anyone ever experienced this before? What could be causing this? Would I still be able to use the gel for western in spite of this? This is very frustrating...
Please help!
You probably over-loaded your wells with protein. What thickness gel and how much protein did you add per lane? It will probably be fine though...
I'm running an SDS gels with proteins samples for western blot and the I noticed the gel is running very strangely. The protein samples have all run together in a straight line! Has anyone ever experienced this before? What could be causing this? Would I still be able to use the gel for western in spite of this? This is very frustrating...
Please help!
Sorry, I cant get you about the "straight line" you meant. Is it after staining or during the running condition? How much time it took to finish the running?
Western blot accupy the very specific antibodies against the target antigen. If you jst want to detect the target proteins in your samples (if is crude lysate) then you may just go ahead. If you want a good profile for documentation then you may troubleshoot and re-run the SDS-PAGE.
It's a 15% gel and I loaded anywhere from 5ul to 15ul of protein in the wells with equal amount of 2X SDS loading buffer. I am not sure about the exact concentration of the proteins which is why I ran the SDS in the first place to estimate but the concentrations should be alright since the post doc had run a gel before me with these same volumes. Should I still move on with the western blot protocol?
I would say yes.
are you staining the gel and seeing the protein bands mingling? or
are you seeing the tracking dye mingling during the run? this is normal.
Hi all,
I am seeing a "straight line" across the lanes during the running of the SDS gel not during staining. The dye seems to have blended with each other on the lanes, the post-doc in my lab says that her gels don't look like that, she has distinct bands running down from each well. I am just running the western for a crude estimation of which sample expresses my target protein the most, so I guess it does not have to be that accurate. I am just concerned that I would not be able to differentiate between the samples when I do my western. Could the samples blend together or is it only due to the dye? In any case, I will proceed and see what happens. However, since I'll be running a lot of westerns in the near future I want to make sure the gels don't act funny, and this "straight line" phenomenon is bothering me quite a bit!
No, it is completely normal. At least for me. It just depends on the volume and amount you're adding per lane. For example, when I run 20+ ug per lane in 10+ uL, the dyes blend together. When I run 5ug or less in 5uL or less per lane, I see distinct dye bands run separately down the gel. I promise that your proteins really do not blend together! The post doc you work with is probably working with larger wells or is adding less per lane than you are.
Quite normal. Try less volume or miss every second well.
Hi again,
Yes, it turns out it is normal after all, I stained the gel to make sure my proteins would come out right. It's the first time I'm doing westerns so I have no idea what to expect and it seems that no matter how many times I read the protocol I just have a million questions and uncertainties. Seems like nothing is going the way it's supposed to. I went ahead and ran another gel, 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 probe with actin antibody, add secondary antibody and then expose the blot and then re-probe with my target antibody? 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 and incubate together. What do the experts thing about this? Also, does everyone use PBS or TBS in their washing/blocking buffer? I see PBS in one protocol and TBS in another. And do we need to prepare the blocking buffer freshly (with milk powder added)? Sorry if I sound frantic because in fact I am!