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.

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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.

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Thanks WAstate

Do you mean that if I want to dot 1uL of sample, I have should dot half of the volume and let it dry then only I dot the remaining half? How do you tell whether the dot was dry or not. From my experience it seems that once you dotted sample it will immediately absorp by the membrane and I consider that as dry. Correct me if I am wrong.



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?

Thank you.

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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

good luck

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