Hello, I performed a WB for CEM's and the run and transfer were fine but something in the blocking is wrong because my ladder gave signal.
-I prepared the PY100 according to its protocol:
http://www.cellsignal.com/products/primary-antibodies/phospho-tyrosine-mouse-mab-p-tyr-100/9411
Dilution for WB 1:2000 and it was mixed with 1X TBST with 5% BSA; (10 ml, add .5 g BSA to 10 ml 1X TBST
-Then, I left it overnight
I washed 3 times and then I used the Donkey Anti-mouse IgG and I prepared it according to its protocol as well https://www.jacksonimmuno.com/catalog/products/109-036-098 .
Finally I prepared the ECL and exposed for 30 sec approximately.
However I did something wrong because my proteins gave a light signal and my ladder gave a strong signal.
Any ideas?
First, make sure you aren't using one of the ladders designed to show up on the blot. Otherwise, if you are using 5% BSA and tween, I don't think the blocking could be ineffective. However, in many years of doing western blots, I've found that some of the molecular weight markers will often show up on the film. I think this is likely because the primary antibody binds nonspecifically to proteins in general and there is a very high concentration of protein at each marker band. I conclude this because I've frequently blotted the same samples with different primary antibodies of the same species and seen huge differences in nonspecific signals. There is also a possibility that the secondary antibody is binding nonspecifically to the ladder. If you want to diagnose the problem, try blotting with a different mouse primary antibody and/or do a blot with just the secondary antibody. You might try diluting the PY100 further or try a shorter incubation at room temperature; often the optimal conditions will vary between labs. Finally, you might try 4G10, which also recognizes pTYR, instead of PY100.
Can you post a picture of the gel? Did all bands in the ladder give a positive signal or only some of them? What is in your ladder?
First, make sure you aren't using one of the ladders designed to show up on the blot. Otherwise, if you are using 5% BSA and tween, I don't think the blocking could be ineffective. However, in many years of doing western blots, I've found that some of the molecular weight markers will often show up on the film. I think this is likely because the primary antibody binds nonspecifically to proteins in general and there is a very high concentration of protein at each marker band. I conclude this because I've frequently blotted the same samples with different primary antibodies of the same species and seen huge differences in nonspecific signals. There is also a possibility that the secondary antibody is binding nonspecifically to the ladder. If you want to diagnose the problem, try blotting with a different mouse primary antibody and/or do a blot with just the secondary antibody. You might try diluting the PY100 further or try a shorter incubation at room temperature; often the optimal conditions will vary between labs. Finally, you might try 4G10, which also recognizes pTYR, instead of PY100.
I agree with Michael's comments above. It can be a ladder designed to show up upon ECL treatment (I used them before) or it is just unspecific adsorption of the secondary antibody over a membrane patch highly enriched in proteins. This is for instance the case of the famous unspecific 60 kDa band showing up when blotting for BSA rich samples.
Reducing the concentration of the secondary antibody should reduce the the unspecific staining of the ladder without affecting specific binding.
If your ladder is blowing out your target's signal and you don't have other ladders, load less ladder on your gel.
Did you attempt longer exposures (> 30 seconds)? Did your target's signal increase if you did?
Did you have a positive control? This may help you determine if your treatments are inducing phosphorylation in your experimental samples.
If your signal does not improve with BSA, you may want to explore using other blocking buffers.
Some ladders use the well characterized Ig's heavy and/or light chain as markers. Therefore, secondary (sometimes primaries too) recognize some MW markers.
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