large proteins don't analyse well-sds page gels - (Jan/16/2007 )
QUOTE (ms-olli @ Jan 18 2007, 04:55 PM)
QUOTE (briskell @ Jan 16 2007, 09:27 PM)
Could anyone help me on this?I am looking at a 106kDa protein in retina. electrophoresis is excellent but only for smaller proteins. I don't get good resolution of high molecular weight proteins, actually I might not even get bands above 70kDa. I have tried different gel concentrations (7-12%)(Tris gels). at 7% the whole sample doesn't run well as if not good homogenisation isdone. at higher percentages I get the previously described motif, so what happens to my high MW ptroteins? do the become fractionated and fall as lower MW?but then the Abd should detect the fraction that has its detection sequence at a lower MW band. do they stay in the membrane during homogenisation and so are located in the precipitant of the centrifugion which goes to the rubbish?
help please help this tissue is hectic. all other tissues work fine.
help please help this tissue is hectic. all other tissues work fine.
How long did you boil the sample with SDS-sample buffer? In the heat there could occure an reaction (i dont remeber well), something like acidic digest of protein. So to long at 100°C and the protein is degradatet...
to digest the protein, one can perform acidic hydrolysis, in HCl 6N, at 100-150°C for several hours.
I can't believe that at pH 7.5 -8.0 you can digest your protein because of 5 minutes boiling.
maybe it's just a problem of extraction/homogenization. I have no experience with retina.
-Missele-
QUOTE (biomaus @ Jan 19 2007, 02:52 PM)
Have done a staining (ponceau red) of you blot? do you see your proteins or any large proteins there? You should use 10% gels (I use them for 95 kd, works well) and let them run not too fast. Do your blotting also not too fast and high (I use 90 mA per gel and 1.25h). I always boil 3-5 min.
my Ponsau shows bands lower than 80kDa for my samples while the marker is fine. I run at 60V until separating gel and then at 100V, then I transfer at 100V for an hour addind 350μl SDS1% for better transfer. this works fine for cortex samples it is my retina samples that do all the mess.
I have also homogenised with ripa lysis and glass homogeniser and I will develop in 20 mins, so I will let you now whether this worked
-briskell-
QUOTE (Missele @ Jan 19 2007, 11:55 AM)
QUOTE (ms-olli @ Jan 18 2007, 04:55 PM)
QUOTE (briskell @ Jan 16 2007, 09:27 PM)
Could anyone help me on this?I am looking at a 106kDa protein in retina. electrophoresis is excellent but only for smaller proteins. I don't get good resolution of high molecular weight proteins, actually I might not even get bands above 70kDa. I have tried different gel concentrations (7-12%)(Tris gels). at 7% the whole sample doesn't run well as if not good homogenisation isdone. at higher percentages I get the previously described motif, so what happens to my high MW ptroteins? do the become fractionated and fall as lower MW?but then the Abd should detect the fraction that has its detection sequence at a lower MW band. do they stay in the membrane during homogenisation and so are located in the precipitant of the centrifugion which goes to the rubbish?
help please help this tissue is hectic. all other tissues work fine.
help please help this tissue is hectic. all other tissues work fine.
How long did you boil the sample with SDS-sample buffer? In the heat there could occure an reaction (i dont remeber well), something like acidic digest of protein. So to long at 100°C and the protein is degradatet...
to digest the protein, one can perform acidic hydrolysis, in HCl 6N, at 100-150°C for several hours.
I can't believe that at pH 7.5 -8.0 you can digest your protein because of 5 minutes boiling.
maybe it's just a problem of extraction/homogenization. I have no experience with retina.
The neutral pH of Tris-HCl at room temperature changes into acidic conditions at 100°C! Than there is a posibility of acidic hydrolysis...But not in 5-10 min
-ms-olli-