Hi,

I never worked with human retina, but 2-3 regions of 8mmX8mm sound enough to me to have a good amount of proteins.

Usually 1-2 rat eyes are enough for what you are looking for...and considering their diameter as around 8 mm... I guess likely 3 pinches in the human are the equivalent.

Are you going to separate RPE from retina or taking alltogether? even in the first scenario you should be able to have a good amount of proteins from both ( with 3 punches).

one strategy I use to increase phosphoprotein level is to perform all the procedures ( dissection and so on) with Sodium orthovanadate buffer.

Hope this is going to help you,

thank you for asking,

Francesca

I really dont know. However, considering the rat retina, I would think that 2punches from the retina would be adequate

Thank you so much Francesca and Rocio. I really appreciate your suggestions.

Hi Srinivas,

there is a simple experimental rule in phosphoproteomics that so if you have the possibility to isolate higher amount of protein, use it because it will increase the number of identifications. I do not have experience with rat retina but I have enriched phosphoproteins from several biological sources. My suggestion is to enrich at peptide level (TiO2, IMAC)because the enrichment methods are more robust than at protein level. I have not used the Pierce kit but I have used the antiphosphotyrosine antibodies and in that case you need a lot of starting material (5-10mg) which for my work was not possible.

regards,

Giuseppe