can u give ur opinion in my western blot results? - (Sep/19/2006 )

hi all

i have a problem that persists for a month now my western blot gives fluctuating results....

so i have some questions:
if i did 8 lanes like that order
1-colored marker
2-postive con 1
3-post control 2
4-sample 1
5-post control 3
6-sample 2
7-sample 3
8-sample 4
and the trnsfer of the marker is ok and after detection the postive control 3 of them are great but the samples nothing at all
so can this be a transfer problem?
another question
after i add sds sample buffer and ddt to my sample and boil it for 5 min i leave them at room temperature and then check by WB may be one or 2 days after does this affeat the results?because i tried one sample after one day and it was ok then i repeat the WB after a week and there was no band at all???
going crazyyyyyy

I think you haven´t attached any image

If the positive control is OK, then, there is no problem of transfer.

I already left some sample in laemmli on the bench a complete week-end. The samples were OK.

yes i didnt attach any pix i just want some answers
so how can i do the same sample twice and gave 2 different results?

my samples are a product of a gst pulldown assay
have anybody anyidea about how long can the gst sepharose beads stay in 4 degree without any loos of the fusion protein?

by the way i dont do elution of my fusion protein from GST beads i keep them in 4 degree and use them for pulldown assay with another candidate protein.

what is the special thing u did with your samples which make them different from controls..reagents may be?...

also check protein concentrations before loading..

another question
after i add sds sample buffer and ddt to my sample and boil it for 5 min i leave them at room temperature and then check by WB may be one or 2 days after does this affeat the results?because i tried one sample after one day and it was ok then i repeat the WB after a week and there was no band at all???
going crazyyyyyy

hi strawberry
my professor said that once we add lam. buffer and boil the sample we can leave it at room temperature foe ever.....

my positive control is my total cell lyate ( conating protein A)
my samples are GST-PROTEIN B and total cell lyate and another protein C

and there is no precipitates in my sds samples.
thankx

you have to boil before to load because some time it can re-oxidize.
But I don't think this is your problem (you would see dimers)

I don't think you have a problem of WB. you have a problem of GST pulling down.
maybe you should write a new post entitled GST pull problem or something like that, to be sure that some GST experienced persons will read your post.