large proteins don't analyse well-sds page gels - (Jan/16/2007 )
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.
do u run a prestained marker along? how does this run in comparison.
the marker runs perfectly this is why I strat thinking that the secret is either in boiling before loading or in homogenisation
This is a protocl I have for homogenising retina. May b this helps.
Lysis Buffer for retina
50 mM Tris 0.605 gms / 100ml
150mM NaCl 0.87 gms / 100ml
1% Triton X 100 1ml / 100ml
+ protease inhibitor.
Add 100µl of buffer for 1 retina in 4 ml of SNAP cap tube.
homogenise it
leave it on ice for 20min to lyse it
15min centrifuge at full speed
aliquot the supernatant and freeze in –80°C
Lysis Buffer for retina
50 mM Tris 0.605 gms / 100ml
150mM NaCl 0.87 gms / 100ml
1% Triton X 100 1ml / 100ml
+ protease inhibitor.
Add 100µl of buffer for 1 retina in 4 ml of SNAP cap tube.
homogenise it
leave it on ice for 20min to lyse it
15min centrifuge at full speed
aliquot the supernatant and freeze in –80°C
thanks so much I will try that out, too and will let you know
Lysis Buffer for retina
50 mM Tris 0.605 gms / 100ml
150mM NaCl 0.87 gms / 100ml
1% Triton X 100 1ml / 100ml
+ protease inhibitor.
Add 100µl of buffer for 1 retina in 4 ml of SNAP cap tube.
homogenise it
leave it on ice for 20min to lyse it
15min centrifuge at full speed
aliquot the supernatant and freeze in –80°C
thanks so much I will try that out, too and will let you know
one more thing, do you do sonication or other ways of homogenisation?
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...
someone in my lab was looking for 270 kda proteins using ripa for lysis and sdspage 3-5%
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...
I have tried different boiling times and temperatures, usually 70-90C for 5min. you mean that the protein could be denatured with heat? well, on this topic I am a bit confused, too. some say to actually boil for 10 min and others to heat but not boil for 5 mins or 7 mins. Others say boiling causes denaturing and others that it helps to reduce the protein and bring it to the its linear structure. I believe it's up to your protein characteristics what works and what doesn't. that's why I've tryied many diffrent conditions and still I can't even get a hint of what the problem is.
what I am afraid of is that such problems are usually caused by something very stupid that you don't even think about...hope this is not the case with me.
Have you done a staining (ponceau red) of your 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.