Hi Gurjot, I cant answer all of your questions, but  SDS-PAGE relies on SDS binding to proteins.

SDS is a C-12 sulfated lipid, so I would be surprised if  lipidated proteins ran differentially on SDS-PAGE because they are already covered with lipid (SDS). 

I don't think the lipidation gets cleaved in std SDS-PAGE. People sometimes see different mobility in SDS-PAGE vs SDS-PAGE/Urea gels, with the lipidated proteins running a bit farther with urea, see papers about Atg8, a ubiquitin-like protein.  

Dear Gurjot

SDS produce negative charge for protein. It seem that SDS can not influenced protein lipidation.

However, using of temperature for protein denaturation can affect the protein-prostethic groups interaction.

You can compare them with running serum lipoprotein in SDS-PAGE and Native page.

Hi Gurjot,

Look up western blots for LC3. This proteins has two forms- one lipidated and another non-lipidated. Although the former would have a higher molecular weight due to the additional lipid moiety attached, it tends to travel further than the non-lipidated molecule perhaps because of the greater solubility.

The lipidation does happen in the cell and is an indicator of the cellular health and physiology. For most people working with LC3, it is important to see both forms as the ratio of the two usually reflects some cellular state so I dont know of anyone who tryies to intentioanlly get rid of the lipidation. I don't know what protein you are working with or why you would what to de-lipidate it but LC3 looks like a good guideline given the non-specific nature of your question.

Hope this helps.

Hello everyone, Thank you for your answers, My main problem is that I have an antibody against a membrane receptor and the sequence the antibody recognises contains a lipidated site (the lipidated site is right in the middle). I already ran a sds-page gel with reduced samples that were boiled at 95 degrees. however I dont see a band. so I thought perhaps this has something to do with the lipidation. I will run today my samples again in reduced and non-reduced conditions, boiled at 95 degrees. could you suggest something more?

Gurjot

Hi Gurjot,

Membrane proteins are a delicate bunch and heating it to 95 c might be the problem. The temperature might be denaturing the protein. Try doing a native gel.

What kind of system do you use? If you are using the NuPAGE gels and the NuPAGE loading and reducing buffer, I'm sure you can just let the sample sit on the bench at room temperature (15-25 c depending on the season and where you are) for 30-45 min to get awesome 1D separation.

Thank you very much for your answer, I am using std lamelli system. and I dont have nupage system here. but i will definitely try by reducing the temp and also native conditions. fingers crossed. but one question:  my protein pi (theoretical) is 9.1. wouldnt that be a problem in a native gel?

Well if you're doing a 1D gel pI is irrelevant but if you're doing 2D, just use a narrow range 6-11 strip and you should be fine.