protein gel electrophoresis - (Oct/29/2008 )
i'm new to laboratory work and research so i'm probably doing some stupid mistake, please please point me to it
Higher bands on my gells are fine and focused but the lower band are not; they look more like cubes and not like stripes. i don't mean the diffuse bands seen on ECL when the sumple is overloaded. even the weak bands on ecl still wide smeared and look like cubes.
can you show us a picture of the gel?
it is hard to troubleshoot without seeing it and without knowing the protocol.
What percent gel are you using? what band sizes are smearing? and how fast (what voltage) are you running it at?
Here is the picture of my gel as you can see the bands at the bottom are very wide and not concentrated, i get such a thing all the time .
The gel concentration is 10%, band size is 18kd , the voltage 100V for 90 min .
There is probably some scientific explanation to this and i'm really looking forward to read what are you thinking of it
this may be something that i have occasionally seen. if it is then it is buffer related.
i have seen, while the gel is running (you have to look closely), a double buffer front and diffuse tracking dye (bromphenol blue) migrating through the gel. if we stop the gel early then we see the same kind of effect that you are seeing(?).
to avoid the effect, we allow the gel to continue to run until the fronts merge and the tracking dye compresses (stacks?). sometimes it looks like the dye "jumps" from one part of the gel to a lower part. after the band sharpens, we allow it to run to the end of the gel (we use a primitive apparatus and use a "plug" gel to seal the plates, the gel run is stopped when all of the tracking dye enters the plug gel at the bottom) and get sharp banding at the bottom.
If you want to look at 18kda you should really go to a higher percentage gel. 12% or 15%
Thanks for the replies, it is really great to get some help,
The problem is that the[b] tracking dye (the blue) does not condens to thin line and i know it should normally , it runs through the gel like 1 cm wide the entire running time. May be that is the real problem. I thought of making the stalking gell bigger, or make the voltage higher when the dye is aproaching the level of my protein. May be the problem is with the sample preparation but i don't know what
Try making up fresh running buffer.