I am trying to determine the in vivo redox states of several stromal proteins in Arabidopsis chloroplasts by labelling them with MAL-PEG and then running on an SDS-PAGE. MAL-PEG binds to reduced thiols in proteins, increasing their molecular mass so that the reduced and oxidised forms of proteins can be separeted on a gel. I extract my proteins by TCA precipitation (which preserves the in vivo redox state). I have 4 different treatments for each sample: 1: DTT, then MAL-PEG --> fully reduced control. 2: NEM, then MAL-PEG --> Blocked thiols (by NEM) control, no MAL-PEG should bind. 3: NEM (block thiols), then DTT (reduce disulphides), then MAL-PEG --> in vivo oxidised forms bound with MAL-PEG. 4: MAL-PEG only --> in vivo reduced forms bound with MAL-PEG.
Now, treatments 2 and 3 work great and run nicely in SDS-PAGE. Treatments 1 and 4, however, are giving me headaches. They are the most "MAL-PEG-heavy" samples, which seems to cause them to aggregate and not run properly in SDS-PAGE. I get almost no signal from those samples with my antibodies, and when I stain my membrane with coomassie, there is indeed very little protein on the membrane for those samples. I am not sure if the proteins aggragate on the gel and can't get past the stacking gel, or already earlier in the tube.
I ran 15% polyacrylamide gels with 6M urea, and also tried 12% gels and loaded less sample to see if the looser gel would help, but it made no difference. Now, the 2 working treatments alredy do give me nice results but it would be nice to get all 4 working to have good controls and more conclusive results. Any suggestions?
Try to run a native gel, I'm no expert with the redox PEG-probe but there are reports that SDS and PEG interact and will smear the gel. Sounds like an interesting project. Good luck!
Thanks for the suggestions! I haven't tried running these samples on native gel, but regular SDS-PAGE should work too (atleast it does in the method paper Peled-Zahavi et al. (2010) Methods in redox-signaling by plant thioredoxins, and I'm using basically the same protocol described there). I will try a 10% gel next, but switching from 15 to 12% made no difference whatsoever...
Hi Lauri,
I was wondering if you found a solution to the protein "disappearance".
I am facing the same problem with Mal-PEG(5000) labeling of a multidrug transporter.
I have tried different buffers (NaHepes, Tris, KPi), pH (7.0 to 8.0), incubation temperatures (0.5-3 hours) and temperatures (0-37 C) but haven't solved it yet. I run on 12.5% SDS-PAGE.
Lauri Nikkanen and Elia Zomot,
If you would like to study the enzyme in their original form, then o for Native PAGE, if you are only interested in knowing the various components of an enzyme then try SDS-PAGE with various buffer combinations and various concentration of proteins while loading the sample and also different percentage of acrylamide.
Hi Elia,
No, I haven't solved the problem, I'm thinking that when whole cell lysates are used and labelled with MAL-PEG (I'm using 5000 as well) without blocking any free thiols (my 1st and 4th samples), so much MAL-PEG is bound that the proteins clump together in huge aggregates and fail to migrate on the gel, be it 10 or 15% acrylamide. I have pretty much settled to using my 2nd and 3rd samples only, where free thiols are first blocked with NEM. These samples run just fine on the gel and also are sufficient for me to get a reliable result from the assay. Labelling without first blocking might work just fine with purified protein or even bacterial cell lysate, but at least for plant total extract I just can't seem to get it working. Perhaps it might work if you use really low concentrations of protein, which might be ok if your protein of interest is very abundant in the cell.
Thing is I need to do Mal-PEG labeling to see if the cysteines are accessible under native conditions and to what degree. Disappearance problem is with samples treated with Mal-PEG both under native or denaturing (1% SDS) conditions. Samples treated with NEM prior to Mal-PEG are just fine, but I still need to be able to see if there are any bands with higher molecular mass.
I've had this issue with different (single or double) cysteine mutants of my multidrug transporter, both in the purified/solubilized form and in the membrane with total proteins. So I should only have one or two Mal-PEG groups bound to every protein. I used 2.5-5 mM Mal-PEG and total protein conc. of 0.5-1 ug/ul (upon labeling).
I'm having similar blotting problem with AMS-albeled total protein extracts from Arabidopsis. I'm running 12% SDS-PAGE (non reducing) and the samples run fine on gel but, as I noticed by Coomassie staining after blotting (1h / 100V/ in cold room) the proteins are still on the gel, failing to be transferred on the membrane. Any idea?Thanks in advance
How manycysteines have your protein? maybe it have many cysteines in the reduced form so in sample 1 and 4 you introduce many malpeg residues increasing enormously the protein size and avoiding the transfer.
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