This is interesting. If I would be doing this, I would FIRST add PI before doing anything else. The idea of their use is to STOP any possible adverse proteolytic action and PI should be added immediately after sampling, not to mention that your sample is the prototype of abundance of enzymes. Centrifuging itself brings proteases in vicinity of their targets and helps fragmentation and lysis. If effective, this will drop down the size of fragments to oligopeptides and less and you will see decreased concentration on Bradford (and very likely increased on UV at start, but then it will go down like a rock as they work). Also, EDTA is excellent controller in 2-5 mM, but not more than 10 mM final. After that it does make strange things and if significantly higher it affects Bradford assay. I never trusted ready made things. Make your own EDTA as a stock (it takes time and requires patients to adjust pH, but it is worth doing). Calculate exactly how much you need to add and do not go over 10mM.  If you have any Ca++ dependent aggregation/polymerization of mucinous proteins, you may see all kinds of things dependent on the way of treatment of sample. Do 0.22 um filtering to get rid of bacteria and debris after adding PI and from there you go further. At least, I will certainly do that. I wish you all of the luck. Your samples are certainly a nightmare to work with, I can tell you.

Dear Ronny,

Since the same sample is giving different concentrations when filtered/ centrifuged, one possible explanation could be : all of the protein is not being centrifuged. To test this hypothesis, try centrifuging your samples at a higher force and if your concentrations increase, you have your answer.

Regards

yes you should try ultracentrifugation for Long Duration

Thanks for replies.

Filtering and centrifuging were measures to get rid of cellular fractions and debris. After that i'm working with the supernatant. Would your suggestion be the same when considering this? Unfortunately i do not have (easy) access to a ultra-centrifuge.

Furthermore the main riddle is why the concentrations change in both directions after adding protease inhibitor. Seems unlogic so far. Maybe i am missing something there.

well are you sure that These concentration values do correspond to Protein of interest

you can always try both the saples for WB first. and i strolgly suggest to check the Profile first. and what filters do you use?

Two possibilities:

1.protease inhibitor is more active in one sample than the other

2.filtration concentrates the protein more than centrifugation

This is interesting. If I would be doing this, I would FIRST add PI before doing anything else. The idea of their use is to STOP any possible adverse proteolytic action and PI should be added immediately after sampling, not to mention that your sample is the prototype of abundance of enzymes. Centrifuging itself brings proteases in vicinity of their targets and helps fragmentation and lysis. If effective, this will drop down the size of fragments to oligopeptides and less and you will see decreased concentration on Bradford (and very likely increased on UV at start, but then it will go down like a rock as they work). Also, EDTA is excellent controller in 2-5 mM, but not more than 10 mM final. After that it does make strange things and if significantly higher it affects Bradford assay. I never trusted ready made things. Make your own EDTA as a stock (it takes time and requires patients to adjust pH, but it is worth doing). Calculate exactly how much you need to add and do not go over 10mM.  If you have any Ca++ dependent aggregation/polymerization of mucinous proteins, you may see all kinds of things dependent on the way of treatment of sample. Do 0.22 um filtering to get rid of bacteria and debris after adding PI and from there you go further. At least, I will certainly do that. I wish you all of the luck. Your samples are certainly a nightmare to work with, I can tell you.

dear,

filtration and centrifugation have different results in proteins and nucleic acids concentrations from the same sample due to the principles involved. if you are adding PI after the procedure, check if there is a precipitation of proteins or debris in your sample (sometimes is difficult to look at them, since it is very small) and them, in Bradford analysis, the concentration will decrease. 

WOW...Too weird?

Let me get this right: you take two identical samples and one you centrifuge and the other you filter through a 0.4um filter... then you set up a Bradford on these and get a number for each (and you have less protein in the centrifuged sample than in the filtered... this part doesn't bother me so far, and in fact makes sense as you would expect protein aggregates to make it through the filter that would spin down in the centrifugation step). Then from this point on you simply add protease inhibitor to both samples at the same amount (and nothing else) and you redo the Bradford.... and when you do this the results for the centrifuge sample go down from the original results, and the results from the filtered sample go up (relative to what you previously got on the same samples)?

If this is the case I am completely stumped? One thing that was said above that I completely agree with was Nebojsa... get your PI into your samples as soon as possible in your procedure... they should definitely have been in there before the centrifuge and filtration steps (although you would have never discovered this very strange phenomenon if you had done this originally). I am very curious to see the results when you finally figure out what is going on (as we use these same PI pills as you)? Have you talked to the Roche tech support yet?

Good luck

Ron

I was thinking about how this could be possible and I have a theory (this is only a theory obviously and doesn't have much basis in reality): I think that when you add the protease inhibitor it affects the Bradford assay. this causes you to get a lower answer for your centrifuged sample, but in your filtered samples you have a bunch of protein aggregates that are making it through the 0.4u filter, so you are originally highly underestimating the amount of protein in this sample in your original Bradford as it is not detecting the aggregates. When you add the protease inhibitor I am guessing it is causing these aggregates to come apart, so now the Bradford although less efficient with the PI added is seeing a lot more protein then it was in the original sample. 

Again this is only conjecture at this point, but is the only reasonable thing I could come up with why the addition of PI to two samples that were otherwise identical in composition to give completely opposite reactions to the addition of the PI in the Bradford assay.

Cheers

Ron

You did not specify how many determinations were done per sample and how different the protein concentrations were. Also, assuming the differences in protein concentration were small, another possible source of variation could be the volume of the samples after centrifugation / filtration. 

Thank you all very much for the responses. There are some very interesting aspects being mentioned. This issue is not off the table yet. I just had to take a little time out to study for an important test. I will proceed with the work next week. And i will definetely keep you up to date.

Also i will give some explanations why i did what and how (and so on)

Thank you so much. You are great