Hi Shahid,

Edoardo is correct.

You are analysing the total cell protein - both soluble and insoluble -  by resuspending the cells in Laemmli buffer. The Laemmli buffer has SDS (2% final in 1x) which will both lyse the cells and solubilise insoluble proteins. 

To analyse the soluble fraction you first need to lyse the cells in a way that will not solubilise the protein - freeze thaw and sonication are other methods - then fractionate the soluble from insoluble proteins by centrifugation. You then add Laemmli buffer to both the fractions and run on a SDS-PAGE. 

This will tell you if your protein is soluble or not.

-Richard

Did you boil your samples before loading on SDS-PAGE?

Yes samples were heated in water bath for ~5 mins at 80 degree centigrade 

i think you miss a step. In order to be sure to have a soluble protein you need to lysate your culture (lysozime 1mg/ml on ice 1hour) and centrifugate it at 31000 x g. then use an aliquot of surnatant for SDS-PAGE. if you find your band you can say that your protein is soluble.

I can see my protein of interest in the lysate run on SDS-PAGE. Cells were lysed by brief vortexing.

Hi Shahid,

Edoardo is correct.

You are analysing the total cell protein - both soluble and insoluble -  by resuspending the cells in Laemmli buffer. The Laemmli buffer has SDS (2% final in 1x) which will both lyse the cells and solubilise insoluble proteins. 

To analyse the soluble fraction you first need to lyse the cells in a way that will not solubilise the protein - freeze thaw and sonication are other methods - then fractionate the soluble from insoluble proteins by centrifugation. You then add Laemmli buffer to both the fractions and run on a SDS-PAGE. 

This will tell you if your protein is soluble or not.

-Richard

Hi there,

Your experiment is only telling you about the level of expression of your fav' protein. For solubility you need to lyse the cells Under non denaturing condition and then fractionate the lysate (usually centrifugation is enough) into soluble (supernatant) and insoluble (pellet) fractions before Laemmli treatment and SDS PAGE.

Hi Dominique,

Thank you for explaining it. I got what i am supposed to do. 

Thank You everyone for the discussion.

Just saw this post.  Glad you now understand the process.  One additional comment regarding successful cell breakage.  Be sure you look at the homogenate under oil immersion microscopy with phase contrast to see if over 90% of the cells are broken.  If you have a significant amount of unbroken cells in the pellet and the protein is soluble then you will have a false positive for inclusion bodies, right?  When you lysed your cells in Laemmli buffer it must have become quite slimy from DNA and was difficult to load into the SDS-PAGE wells.

I didn't quite get that. If significant number of cells are unbroken in the pellet...

When i centrifuge post sonication with lysis buffer containing no denaturant (No 6M guanidium or 8M urea) and collect lysate isn't it supposed to be free of inclusion bodies and hence contain no insoluble protein fraction.

Regarding lysis in Lamelli buffer, it does intact turn a bit slimy but we high speed centrifuge it and load the supernatent in the gel. Supernatant is just fine.

I understand what you mean. Some part of my target protein shall/could remain within the pellet and not come in supernatant due to the fact that all cells won't lyse (I hope thats a very minute fraction). In subsequent experiments I have used lysozyme for cell lysis and also done sonication prior to centrifugation for collection of lysate. I ran the gel for lysate only and saw my protein of interest in the total cell protein fraction. I didn't however, lyse or run the pellet. I will however run the pellet too next time. Thank You for you input.

Regards