retina homogenization for western - (Dec/04/2006 )
the lysis protocols I have already used are not giving me a band at 100KDa, while the 7% & 8% gels don't analyse my homogenized samples and the 10% gel gives me a very pale or very absent band. Any ideas why this is happening? the antibody is working fine on other tissues and the band is not getting stronger no matter how dilute or concentrated the antibody is.
Are you sure that you are loading an enough amount of protein? have you done a Bradford or similar?
I have loaded different concentrations of protein and I get no signal below 80-100 μg of protein loads. retina is giving me a 
headache since it doesn't run well in electrophoresis when it is concentrated and when dilute there there is not enough space in the wells to load greater volumes of samples.
My thought is that homogenization is not good and I am looking for a good protocol, and as you probably know articles are not very clear on all the steps.
headache since it doesn't run well in electrophoresis when it is concentrated and when dilute there there is not enough space in the wells to load greater volumes of samples.My thought is that homogenization is not good and I am looking for a good protocol, and as you probably know articles are not very clear on all the steps.
Very strange
I don´t know how retina is, but could this sample have too much lipids so it doesn´t run well? What about precipitating proteins so you could have a purer sample? By the way, you can try asking the authors your doubt with published protocols. Sometimes they ignore you but others are kind
if ur protein is expressed in only 1 layer of the retina, then u might have a difficult time detecting it.
we used to use 7% gels for retina samples.