Western not working anymore - (Jul/22/2010 )
sera_tonin on Fri Jul 30 19:51:42 2010 said:
cm13 on Thu Jul 29 08:44:13 2010 said:
Hello again.
So i retried my westerns as before, but this time i washed 4 times for 6 minutes in TBST where needed.
I did see an improvement in the clearness of the blots.
I also ran a range of different protein concentrations (5, 10, 20 and 30ug per well) just to see if the amount i was adding made a difference.
The blots appeared clearer after the extra washings.
With the blots where i did a 1/2000 or 1/5000 dilution of the Primary antibody, nothing appeared.
With the blots with a a 1/1000 Primary concentration, there were bands, but they were non-specific.
They were stronger (specific and non-specific) bands in the 20 and 30ug wells, but that would be expected.
Im going to try a 1/1000 of primary and a 1/5000 or 1/10000 of secondary next, but does anyone have any other suggestions of what to do to avoid non-specific binding?
nonspecific bands are usually from too much primary antibody, in my experience - but if you're not getting bands at a higher dilution, then you don't have much choice. it could just be that this particular antibody has nonspecific binding - have you used it before without getting the extra bands?
Yes. I tried it before and it got specific bands. But to repeat the experiment a while later gave me non-specific ones.
I thought it could have been because i re-use antibodies a couple times, but even with fresh antibody, the same thing happened.
Could this mean i have a problem maybe with my cell lysates?
How is your antibody stock stored? If it is in the fridge, then it may be going off. All our antibodies get stored aliquotted at -20 and are only thawed once to prevent degradation. I have come across antibodies that degrade very fast (less than a week in the fridge).
bob1 on Sun Aug 1 22:15:31 2010 said:
How is your antibody stock stored? If it is in the fridge, then it may be going off. All our antibodies get stored aliquotted at -20 and are only thawed once to prevent degradation. I have come across antibodies that degrade very fast (less than a week in the fridge).
I freeze it in its diluted (in BSA) form at -20C.
I also only ever re-use the antibody 3 times at most, as it would go off afterwards.
cm13 on Fri Jul 30 19:56:07 2010 said:
Yes. I tried it before and it got specific bands. But to repeat the experiment a while later gave me non-specific ones.
I thought it could have been because i re-use antibodies a couple times, but even with fresh antibody, the same thing happened.
Could this mean i have a problem maybe with my cell lysates?
did you prepare the tissue the same way as before? it could be something like, this time you solubilized more membrane proteins than last time, and some of those membrane proteins are binding the antibody. i don't know
sometimes the biochemistry gods conspire to throw a monkey wrench into our experiments, for no logical reason... Hi again everyone.
At the recommendation of others, i performed westerns with the following concentrations:
Primary 0, Secondary 1:1000
Primary 1:1000, Secondary 1:1000
Primary 1:2000, Secondary 1:1000
The results were as follows:
Primary 0, Secondary 1:1000 - Bands appeared as before - as if the antibody attached to a lot of proteins
Primary 1:1000, Secondary 1:1000 - Same as the blot with no primary, but with some bands being stronger.
Primary 1:2000, Secondary 1:1000 - No bands at all.
Surely this indicates that im having a problem with my Secondary?
Should i try 1:1000 for primary and then lower concentrations for the Secondary?
you can try or you can preabsorb your secondary with your sample.