Manual Dot Blot and SA-HRP detection Protocol...HELP - (Feb/09/2007 )
Hi to all members,
I need a protocol of manual dot blot..can anybody help..pls provide the protocol as detail as possible. I have tried to use dry and wet membrane but the spots were still blotchy.
I am using SA-HRP for detection. What is the best washing buffer to be used? Pls describe the method in detail.
Thank you very much.
I think the dot size depends on your dotting technique or how much and what you use to dot your samples. I use a pipet tip and 1-2 uL. If you dot slowly by letting part of your volume dry first, the spot will not grow out of control. I used the protocol that you can find on Abcam's website in their protocol section, and it worked just fine. If you are using SA-HRP, then you need a biotin-conjugated primary antibody or an unconjugated primary antibody plus a biotin conjugated anti host species step in between. Do not use milk in your buffers if you are using biotin...it helps with background, at least for me. I used TBST for washing and BSA/TBST for incubating steps.
I am using Denhardt solution for pre/hybridization. How long did you wash after hybridization? You used TBST for washing after the hybridization steps? I use SSC/SDS. What detection method did you use? How long did you wash after detection steps?
i have done dot blot 2-3 times only.....none of my labmates has experienced with it , so i just follow the blotting machine manual and read some book and i did good.
i make my sample of desired conc and add it in sample well with buffer and blotted.
washing was just same as the southern or northern