How to normalize protein vs its phosphorylated form? - (Feb/27/2008 )
Hi every body!
I'm performing a Western Blotting of Smad proteins and their phosphorylated form after a treatment. I haven't obtained any differences between the phosphorilated forms with that treatment, but there are differences when I use an antibody for the total Smad. Must I normalize the loading according to the total protein levels?. Can any one help me please??
Thanks in advance for your responses.
Hello,
One possibility is that it is also your protein amount that changes upon treatment and not only its phosphorylation. In this case, I would suggest you to pick a protein that you know is expressed and unlikely to change due to your specific treatment and use this as a loading control (tubulin, b-actin are common ones, but do make sure they are not likely to change in your experiment!). Alternatively, you can stain the blot with Ponceau or Coomassie (if you never want to use it again for immunodetection) to show equal loading (however, I don't think it is a very elegant way and referee's can ask you to present an immunodetection loading control especially if the change you are showing is quite subtle).
On the other hand, I'm not sure your message describes the above case. Do you see no-difference in phospho-bands and difference in total Smad? In this case, the phosho-ab can be crap. Are you sure it detects the P-Smad and not a non-specific band at about the right size and are you sure about the affinity of the ab?
An other alternative in this case could be epitope-masking due to your treatment. Can you try alternative antbodies and/or can you check which region the ab detects and whether it can be affected by your treatment? Do you see band-shift after treatment?
Do you use a general Ser/Thre or Tyr phospho ab or a specific one to a particular Smad phospho-site?
I would definitely try and sort out the protein amount first making sure whether the amount changes or not and then address the phospho-band question.
I hope you can figure it out quickly what is going on.
Krisztina
ps if you still struggle with it, it could potentially help us give further ideas of we saw your images and/or more detailed description.
I'm performing a Western Blotting of Smad proteins and their phosphorylated form after a treatment. I haven't obtained any differences between the phosphorilated forms with that treatment, but there are differences when I use an antibody for the total Smad. Must I normalize the loading according to the total protein levels?. Can any one help me please??
Thanks in advance for your responses.
I agree with Krisztina. Phospho-specific antibodies can be tricky to work with and more times than not it turns out that they are not identifying the specific protein but a band at the expected size. First things first, if you are trying to compare expression levels of anything in one lane to another, you MUST do a loading control. Do an actin or tubulin western to show that each lane has the same total protein. This is the only way you can say that the total Smad changed rather than the amount loaded into each lane was different. Next, treat one sample with a phospatase and see if the band that you think is phosphorylated Smad disappears. If not, sorry but your antibody isn't good. If you have purified protein, try to phosphorylate it with a kinase known to have Smad as a substrate, run this and see what happens to your phosphorylated signal (it should be very strong). Otherwise, if you have the purified protein you can do an immuno-absorbtion (incubate protein and antibody). If this also causes a loss in signal it shows specificity of the antibody.