western blot: why are proteins not separated? - (Dec/04/2008 )

hi everyone,
i did western blot today.
my protein lysates were collected using sample buffer,
and was boiled at 95`C for 5 min before keeping it at -20`C,

after protein quantification, i was to run SDS gel for the protein today,
but the protein bands did not run down the gel.... not even the stacking gel (upper)....
wat can be the reason??
i used 100V for stacking gel, i noticed that the ampere indicated just 2 or 3....
i know this is not normal, but i also prepared new 1X TG SDS running buffer.....
result was the same....

not boiled enough?
i noticed that there are still some small, very tiny white substance in the protein lysates...

oh, NO!!

Hi,
your electrophoresis apparatus short-circuited. The electric current has to run through the gel for the protein separation to occur. If the electric current passes though the buffer bypassing the gel, your proteins will stay where you loaded them, except for randomly diffusing over time. Did you check this? Sometimes, adding too much buffer can cause this.

oh...

didnt realize abt short circuit, i used abt 300 ml of sds running buffer...
will check again
thanks

It might be possible that your power supply apparatus is not working. The fact that you are getting only 2 to 3 amps in spite of setting a large voltage shows that no electricity is passing through the gel. The amps (short for amperes) is the measure of the electric current, while the voltage denotes the potential difference between the electrodes. Therefore, it is the amps that tell you truly how much electric current is actually passing though at any given moment.
Try using a different power supply. It might also be possible that your gel apparatus has something wrong with it's wiring. Check the electrodes and wiring.

sorry if this is insulting but...

did you put buffer in both the top and bottom of your apparatus?

is your buffer prepared properly? check the recipe and compare to what you made (if the pH is too low then you will get little to no migration).

you may have over boiled your sample. those white specks could have been aggregated protein. large aggregates won't migrate. you can incubate your protein in loading buffer at 65C for 10-20 minutes before loading.

the white could be precipitated sds. if you have kcl or cacl2 or some other cations present you can make an insoluble salt of sds (no longer sds, maybe kds or cads...). you should still see some migration but a lot of streaking.

finally, what is the concentration of acrylamide in your gel (and the size of your proteins)? the pores may be too small for your proteins (or your protein of interest, at least) to migrate through.