Miroslav Perniš

Slovak Academy of Sciences | SAV · Department of Reproduction and Developmental Biology

Hello, I have been trying different approaches to purify the „secretome proteins“ from my suspended cell culture media. I have tried few protocols and developed one combining them, that gave me quite pretty results. But I wanted to improve the purity of the proteins so I treated the medium with PVPP to remove phenolic compounds. (shaking for 45 min., 4°C). I have done this before, but now I have used a HCl-tretaed PVPP (Charmont et al., 2005). The PVPP was boiled with 10% HCl for 10 min., rinsed with water untill neutral pH, dehydrated with acetone and grinded with mortar to powder) This should increase polymerization and remove metal ions and contaminants from PVPP. Then I proceeded how usual with protein precipitation, washing etc. ...to SDS-PAGE. But it came up like you can see on the picture 1. Bottom of the gel is not ok either, but the upper part is a disaster. Could it be that I have rinsed the PVPP poorly and there were still HCl remains that acidified my proteins? Could it lead to this? Or do you guess any other reason?

The picture 2 is to show we did not have this problem in former runs. This gel was only a try-out for protein load and there is low proteins loading. But it is much better than the new one.

I will be gratefull for any thoughts.

Miro

Hello,

a brief background ...We'd like to analyze pine secretome of different lines obtained from SCC media. After desired cell growth, the media is filtered, checked for potential intracellular contamination, then concentrated using Amicon Ultra 10kDa cut-off. Firstly we tried buffer exchange on Amicon Ultra after ultrafiltration and then direct use for SDS-PAGE, but the gel quality was low. Clearly, the samples needed to be purified way more. We decided precipitate and wash the proteins. We have used different approaches (TCA/aceton precipitation, TCA/aceton wash, ammonium acetate in methanol precipitation and consequent washes). Every variant included the sample mixing/incubating with ethyl acetate to remove possible secondary metabolites and then with PVPP to remove polyphenolic compounds. The pelets were disolved in proper buffer and the SDS-PAGE was performed. The quality of the gels was still unsatisfactory and we suspected there was also an interaction with protein assay as we did not get equal sample loadings (notable in the lanes in the gels) with any of two assays (BCA and Bradford).

Then we found protocol from Wang et al. 2006 who used it also for pine leaves and tried it out for our samples. Shortly: the proteins are firstly precipitated from the concentrated media with 10% TCA in aceton, the pelet is washed with ammonium acetate in methanol and acetone and then the pelet is resolubilized in phenol/SDS buffer, incubated, centrifuged. Then the upper phenol phase is collected and ammonium acetate in methanol added to precipitate proteins.

PROBLEM: After centrifugation, we got three layers, upper clear, middle white and bottom more or less clear. (see the picture) Problem is, as appeared after precipitation, that the upper clear phenol phase did not containe any proteins, the most proteins where in the middle white layer (which we believe was also phenol) and some remained in the bottom layer. I am pretty sure this is not the way it should be and the white part are some compounds that will still interfere with the analysis. We really hoped we will get rid of them using phenol extraction but they appear to be a pretty tough ones... In addition, we tried also "traditional" phenol extraction - firstly incubation with phenol/extraction medium then precipitation. Same results. Any ideas what could cause this and what may be the solution? It is essential for us to obtain good quality proteins and the same sample loading as it influence the whole analysis.

We appreciate any idea and we thank you all, who may got some, in advance.

Link for the article (hopefully it works):