Seems you have too little protein for the dot blot. Increase the amount if you are able to do so.

@Maria del Pilar Corena-McLeod

but when i load the same onto Gel and seperate it i see a band (see picture). The same protein mass (unseperated) was loaded onto the dot blot

Hi Linda

The principle in Western blot and Dot blot are similar but not exactly the same. In Western blot you perform an electrophoresis previously. This one can be made it in different conditions that may change the conformation and the environment of your protein. For example, you can perform native or desnaturalizant electrophoresis, under reducting or non reducting conditions and in this way you may obtain different results. On the other hand the separation obtained by the electrophoresis help to "clarify" your crude sample and maybe this one be the reason because you can see the band in WB.

In dot blot, you put the whole crude sample and maybe highly hydrophobic proteins present in it does not permit your protein binding. May be steric hindrance or hook effect for too high concentration in the crude sample does not permit the detection in dot blot .

Anyway, I don't be clear about your dot blot results. I see positive spots in the membrane and why to change the streptavidin if it works well in WB? I dont think that this option result.

Best regards, Oscar