If you are here, you have something strange going on with your western blot reagents indeed, so lets jump right in.
By now you have confirmed that Step 1 of our blank film troubleshooting protocol worked but somehow you ended up with no dots at the end of step 2.
Step 2 contains 3 variable steps that we now need to address methodically.
Those variables are:
- Time x 2 incubations
- Blocking buffer in Step D
- primary antibody
So go back to step 1
A. Pipet 1ul of your secondary antibody directly onto a piece of membrane (nitrocellulose or PVDF)
B. Pipet lul of a secondary antibody you know works as a positive control onto the same membrane (too clarify you should confirm with a lab mate or someone in a nearby lab they have successfully used this secondary in the last week)
C. Draw a circle around the two spots with a pen and label them
D. Block the membrane in blocking buffer of your choice for 30 min at room temperature (RT).
Stop – Develop the dotblot with your ECL reagent and film/machine of choice.
2 dots on the film- Congrats! the problem is not your blocking buffer - move to Step 2
No dots on the film Something is strange about your blocking buffer. Try different variables on blocking buffer until you get 2 bands at this step (BSA, milk, FBS, or none).
Step 2 – Repeat Steps A-D then
E. Add the Secondary Antibody in blocking buffer to the membrane for 30 min at RT
Stop – Develop the dotblot with your ECL reagent and film/machine of choice.
2 dots on the film- Congrats! the problem is not your blocking buffer, incubation time, or secondary blocking binding in some bizarre way - move to Step 3
No dots on the film The only possible explanation is something is strange about your secondary antibody, time to buy a new one.
Step 3 – Now repeat steps A-E then
F. Wash the membrane X 3 changes in TTBS
Stop – Develop the dotblot with your ECL reagent and film/machine of choice.
2 dots on the film- Congrats! the problem is not your TTBS.
No dots on the film The only possible explanation is something is strange about your TTBS, make it fresh and test again.
You should be ready to go back to the troubleshooting protocol and repeat Step 2.