western bands look funny - (Jun/21/2010 )
I am attaching a recent western blot that I obtained from both the nucleus fraction. I am wondering if you could help me understand of why my blot looks the way it looks right now. Mainly I am wondering of why the shapes of my protein is not the nice squares that I mostly see in publications? My actin bands (the bottom bands) are oddly shaped and sometimes it is longer in some lanes and shorter on another. Consequently I find it difficult to tell if I am getting the same loading on all my lanes. Another issue is those other faint bands between the top band (which I detected for) and the bottom thick actin band, that I think is because my primary antibody concentration being too strong thus the non specific binding. I hope someone can echo my interpretation of the blot.
I have spoken to few people and some told me that upon loading my samples I should run it quickly, otherwise I would be seeing those 'sticks' at the end of the bands. Another told me to make sure that my gel is set prior to loading, but I think I am sure the gels are set, since I left the remainder acrylamide solution outside and watched that one to solidify before confirming that the gel is ready to use.
What I am trying to use the blot for is to see difference in relative intensity of the top thick bands, and hoping to eventually semi quantify them. I am thus wondering if these odd shapes will have effect when scanned using densitometer and quantified with the appropriate software?
Thanks so much in advance for any response!
You blot is fine... those ones you see in papers are "perfect" ones done as repeats of experiments to get perfect looking pictures. People often also chop off nonspecific bands.
The faint bands you see below your protein of interest (POI), may not be non-specific, they could be modified forms or incompletely translated forms of the POI.
If you are worried about the gel not being completely set, leave them overnight at 4 deg C in a moist environment (wrap in wet tissue paper + cling wrap) and they should be completely polymerised. They also set more evenly at 4 deg C if you want to do the whole thing in a cold room.
Changes in size of band may have something to do with polymerisation of the gel, but it could also have to do with slight differences in lane width from the combs, incomplete mixing when pouring the gels, salt concentrations in the gel, uneven electrical field in the tank... etc.
Quantitation is not a reliable technique for westerns in my experience, minor variations in the way the gels are run, loaded, buffer composition, voltage variances, pretty much anything really affect the results you get from the quantitation. I'm not trying to dissuade you from using the technique, but it is something to be aware of.
Thanks Bob1. There are two things that I'm trying to change now. One is to increase the length of the stacking gel. And second is to increase the padding in the transfer unit. I hope these two can help to increase resolution of the bands.
let us know how it goes!
So to update you folks that have kindly helped me when I was desperate with my blots, here are several things I changed that seems to work to improve my blot to look more like those pretty blot in the literature.
1. I bought a new electrophoresis and transfer system. It cuts my heart ache by half.
2. I did increase the padding in the sandwich by adding a layer of filter paper to ensure tightness.
3. I corrected the length of my stacking gel to at about 1 cm (it used to be only few mm).
4. I did load samples quickly and then run immediately.
The solidity of the gel does not seem to be an issue. With the old electrophoresis system however my stacking gels would sometimes web, making loading very difficult and slow, and I figure it was due to uneven tightness in plate clamping. I don't have to deal with this issue with the new electrophoresis system.
ok again, thank you!
Good to hear it is working. Proper stacking gel length can make all the difference.