3 replies to this topic

[joseant78]

Hi all,

I´m having some problems with the resolution of my proteins in the gel. I have to check fosforilation in tagged proteins. It happens very often but not always. The day before I run some proteins nicely and when I do it again, with the same samples, it happens: protein marker is almost invisible in the gel, ponceau after transference is very clear, and I eventually obtain my protein very diffused and irregular when I develop the blot..., that´s why I ask u for help, to identify the possible variable/s. First I thought it was a problem of buffer, so I did some tests with different brands of different products ( glycine, SDS, Tris... Nothing changed. I changed even the water...again I didn´t observed any change,  
My protein is about 150 kD of MW and I use a 10% ABA gel, made with a stacking buffer from Tris-HCl pH6.8 and a resolving buffer from TrisHCl pH 8.8. My running buffer pH is 8.6. I run 2 small gels at constant intensity of 30 mA. First the voltage is about 70v and later on, in the resolving part, it changes to 140v aprox.
I need to resolve this big protein and its phosphorilation bands, so I have to run the gel until the band that corresponds to 150kD is between 2/3 and1/2 of the resolving part, which means that I have to run the gel for 5 hours aprox., allowing the gel to run over 1-2 hours after the blue front  run out. When I try less concentrated gels ( 8% ABA) never resolves the proteins nicer than 10%, and I observe the same problem indeed ( my samples and protein marker becomes abnormally clearer).
The transference is usually made in a transfer buffer similar to running buffer with 20% of methanol( at constant intensity of 400 mA along 40-60 minutes for 2 small gels).
Now the only thing I can do is to observe whether the marker ( I use 5 ul of thermoscientific pageruler) is quite visible in the gel or not...and then go on with my WB, but sometimes I have to do it 5 or 6 times until I get the proper resolution...and the problem is the degradation of my samples with the consecuent waste of time and money)
I measured all pH, even after running I measured the running buffer ( which did´t change from 8.6) cause I believed it was a pH problem. I read maybe it might be a problem with the resistance of the gel, which increases and consecuently would made increase the diameter of pore in the ABA gel and this could produce a protein faint.....

Any comment/suggestion/ criticism/ are welcome, thanks a lot for your help and your patience

[mdfenko]

have you stained the gel (not transferred) to see how the protein resolves? if the bands are sharp (enough) then your problem is occurring during the transfer.

if not then you're having a problem with your sample. how are you storing it between loadings? are you storing in sample loading buffer? frozen, refrigerated or room temperature?

[joseant78]

I didn´t stain the gel yet, I will, ...but it seems to be the running, cause the abnormallity of the protein ladder.
I store my samples at -20 ºC in a  sample buffer made with tris-HCl 6.8 Glycerol, SDS, bromophenol, DTT and water.

[mdfenko]

run parallel gels, one to stain and one to transfer. this will help you determine where the problem is occurring.

when you defrost your samples do you ensure that all the sds is back in solution?

do you clarify the sample to ensure that you only load soluble proteins?