western blot milk dots - (Aug/09/2006 )
Dear all,
recently I found that after I block the blots in marvel milk for one hour the blot has quite lot milk dots on it by looking at the light. originally I thought because of milk undissolve completly. but after I stir the milk for 10 mins should dissolve completely I still see those dots. What is the problem?
anyone has ideas? Thanks a lot
is it actually milk dots? (ie does it wash off with wash buffer?)
i have encountered something vaguely similar, but the white blotches were more to do with inconsistent methanol saturation of the blot post transfer.....all was fine though, so i would proceed as normal.
I actually filter the milk I use for Western through a coffee filter (my boss is too stingy to buy us real filter papers!)... but then again..it should come off after washing, if it were undissolved milk. 
I had also this problem and saw dots on the blots even as I vortexed the milk-solution. With BSA I never have this problems. I think, to filter the solution would be a good idea.
Do you use non fat milk?
I did use marvel non fat milk and i vortex the milk and rotate for a while. now the milk dots are gone, but my blot still has a black back ground. what is wrong? i really really got tired with this staff.wuwu want to cry
Do you use non fat milk?
I did use marvel non fat milk and i vortex the milk and rotate for a while. now the milk dots are gone, but my blot still has a black back ground. what is wrong? i really really got tired with this staff.wuwu want to cry
what is the concentartion of ur antibodies?
how about ur washing step
can u give more detailed?
Do you use non fat milk?
I did use marvel non fat milk and i vortex the milk and rotate for a while. now the milk dots are gone, but my blot still has a black back ground. what is wrong? i really really got tired with this staff.wuwu want to cry
what is the concentartion of ur antibodies?
how about ur washing step
can u give more detailed?
the reason I am confused is that the step I use before I got great blots, just from certain days my blots become poor but others in the lab are still fine so means only something of mine is wrong. I can not figure out, bu t Ｉ ｄｉｄ ｓｅｅ ｓｏｍｅ ｍｉｌｋ ｄｏｔｓ， ａｆｔｅｒ ｉ ｓｏｌｖｅ ｔｈａｔ， ｓｔｉｌｌ ｈａｓ ｂｌａｃｋ ｂａｃｋｇｒｏｕｎｄ． ｔｈｅ ｓｔｅｐｓ ｉ ｕｓｅ ｅｘａｃｔｌｙ ｓａｍｅ ａｓ ｂｅｆｏｒｅ．
Do you use non fat milk?
I did use marvel non fat milk and i vortex the milk and rotate for a while. now the milk dots are gone, but my blot still has a black back ground. what is wrong? i really really got tired with this staff.wuwu want to cry
what is the concentartion of ur antibodies?
how about ur washing step
can u give more detailed?
the reason I am confused is that the step I use before I got great blots, just from certain days my blots become poor but others in the lab are still fine so means only something of mine is wrong. I can not figure out, bu t Ｉ ｄｉｄ ｓｅｅ ｓｏｍｅ ｍｉｌｋ ｄｏｔｓ， ａｆｔｅｒ ｉ ｓｏｌｖｅ ｔｈａｔ， ｓｔｉｌｌ ｈａｓ ｂｌａｃｋ ｂａｃｋｇｒｏｕｎｄ． ｔｈｅ ｓｔｅｐｓ ｉ ｕｓｅ ｅｘａｃｔｌｙ ｓａｍｅ ａｓ ｂｅｆｏｒｅ．
I had similar problems. We found out in the lab that it was because of the secondary Ab I was using: we centrifuged and the background disappeared. It seems that sometimes you can have aggregates in your secondary and that this gives you the high backgroung but you remove it by centrifuging your Ab. I hope it helps
filtering of freshly prepared skim milk is necessary; or use alternatives such as Rotiblock, you can even do Ponceau S after blocking