Author Topic: Protein extraction from culture medium  (Read 17572 times)

daveytea

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Protein extraction from culture medium
« on: February 10, 2007, 05:00:00 PM »
Dear all,

I'm trying to extract proteins secreted into the culture medium by a bacterium. I've tried acetone precipitation method. After the bacteria grow to mid-log phase, I've centrifuged down the cells at 6300rpm for 10 min and filtered the supernatant to remove all the bacterial cells. Next, I've mixed2.5X volume of ice cold acetone (100%) to 1X supernatant and incubated at -20oC with occasional shaking for one hour. Precipitates appear once I've added the acetone. Then after the incubation, I tried to centrifuge down the precipitates at 5000rpm for 5min. To my surprise, the pellet are dark brown slurry substances with little brown crystal-like thing. I wonder if it is protein. I think that protein precipitates should be white to light yellow ppt (i've done 2-D clean-up kit with other protein sample previously and quite sure that the ppt that appears during 2-D clean-up process are way different from the slurry substance that i've got with the aceton-medium mixture). As i'm using BHI medium and i thought that there may be small amount of protein exist in pure medium, so i tried to add acetone to pure BHI medium. Precipitation also occur immediately.  Since it looks quite turbid, i wonder if it's really due to the protein existing in BHI medium as it's not quite possible to have some much protein in the medium. Have anyone done any secretome experiment that used complex medium? I've seen ppl used BHI medium (Infect Immun. 2002 July; 70(7): 3396-3403) and LB medium. But seems like BHI medium is not a good choice as seen in my case... Can anyone suggest what happened?

Thank you so much~

Best Regards,
Dave


rantanplan

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Re: Peotein extraction from culture medium (daveytea)
« Reply #1 on: February 11, 2007, 05:00:00 PM »
Maybe your 2.5x vol. Acetone isn't enough? I use 4fold...
Another point: 5000 rpm = how much g? We have had problems with precipitating proteins using about 10.000 g from time to time - and I dont think you will reach that with 5000 rpm (depending on the centrifuge you use   )

mrmoon

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Re: Peotein extraction from culture medium (rantanplan)
« Reply #2 on: February 11, 2007, 05:00:00 PM »
I think Rantanplan has a point. To spin down protein pellets the g force should be much higher than to spin down cells. You should find out the corresponding rpm that you should apply with your centrifuger to obtain the right xg. Our centrifuger is Eppendorf 5415C. With it, 5000 rpm is not even 2500 xg.
As for the color of the pellets, I have no experience with media for bacteria. I agree that my protein pellets are alway white-ish. The BHI medium does contain some digests which are peptides I think, and protease inhibitors? and some salts. When there are considerable level of salts I don't like acetone precipitation because salts will be co-precipitated. Can't say it attributes to the brownish color though.
I once ran a few media samples from a customer. He span them down to concentrate using concentrating columns, which I believe helped removing smaller molecules. I performed cleanup. The procedure was very normal. The gels came out quite clean with fewer number of proteins than from cells. Maybe you want to try one of the concentrators. Milli-Q has a selection of various sizes.

Ambort

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Re: Peotein extraction from culture medium (mrmoon)
« Reply #3 on: February 11, 2007, 05:00:00 PM »
Ciao,

I also use the concentrators from Millipore. Precipitation did not work in my hands for culture media, because there are too much different salts present. Some of them may form insoluble precipitates. With very big volumes you may not get rid of your salts.

best regards

Ambort


daveytea

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(No subject)
« Reply #4 on: February 11, 2007, 05:00:00 PM »
Thanks all for the reply.

For 5000rpm, it accounts for around 6000xg in our centrifuge. I guess I may try 10,000xg in future.
Actually I'm planning to use the concentrators from Millipore too, just that seems many publications describes the use of precipitation for bacterial secreted proteins, I wanna to try out if this method is workable. I have also tried to do the TCA precipitation method. (add 250ml of 20% TCA to 250ml of medium and incubate at 4oC overnight with stirring, then centrifuge the mixture at 7000xg and wash pellet 3 times with ice-cold 75% ethanol). It did gives some ppt that looks like protein.
The main thing that concerned me most is that the culture medium (BHI or LB) contains proteins that I'm afraid their amount may interfere with my bacterial secreted proteins. In order to verify if there are really a lot of protein present, i've tried precipitation methods with pure medium (BHI and LB). The medium looks turbid once i've added either the acetone or TCA. After centrifugation, the pellet in the once with acetone added gives a brown-gel like substance which doesn't look like protein ppt at all. As with the once that is ppt with TCA, a light brown ppt appears as the pellet and i'm not sure if it would be protein. Just wonder have any of you has been working on BHI or LB medium and did you guys got the same problem. I simply can't understand how can ppl use BHI or LB medium for secreted protein extraction if the protein are complicated with proteins!?

Thanks all again for the comments.

Best regards,
Dave


ejoz

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Re: (daveytea)
« Reply #5 on: February 21, 2007, 05:00:00 PM »
Hi,

I use AB media, and i do experience some form of brown jelly like ppt after precipiation. Wat i do is that i was more times and volumes. afterwards, the finall pellet dissolved in lysis solution then send for ultra-centrifugation then collec the supernantant. My 2D profile seems okie, but proteins maybe lost due to ultre-centrifugation. But never go n verify them. Maybe i will try precipitating just the pure media n take a look.

regards
TJ


Dambort

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Re: (ejoz)
« Reply #6 on: February 22, 2007, 05:00:00 PM »
Ciao,

it is really not that easy to get a nice gel of secreted proteins into media. One main problem is the medium composition itself. You have to make sure that the medium itself you use is free of any protein source used. For example LBA medium is full of proteins and you may not be able to find secreted proteins because they are just to low abundant compared to the medium. Therefore you should select conditions that are protein free before you start your real experiment. Thus you have to find conditions in which your cells grow properly and this a minimal medium without exogeneous protein source. The best strategy would be to find such conditions and you do first ultracentrifugation of your medium to remove any lipid contaminants. TCA precipitation for large volumes will introduce to much TCA that can be removed for successfull isoelectric focusing. Also acetone precipitation may be problematic. There exist many publications on precipitation of secreted proteins from media but unfortunately, the proteins that are identified are most serum-derived proteins from media supplements! To this problem can really circumvented by using minimal media and then ultracentrifugation to remove particles, cells, exosomes, lipids, transmembrane proteins, cell debris usw. Then by using ultrafiltration in combination with three times washing of concentrates with low-salt buffers will remove salts and make your sample compatible with first dimension IEF.

best regards

Ambort


Dambort

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Re: (Dambort)
« Reply #7 on: February 22, 2007, 05:00:00 PM »
Sorry I wanted to say LB medium and not LBA!

Ambort


Dambort

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Re: (Dambort)
« Reply #8 on: February 22, 2007, 05:00:00 PM »
One more thing to add. When I started with media analysis of secreted proteins into culture media supplement with 5% fetal calf serum and did precipitation of this, then the 2-D gel looked like a classical human plasma 2-D gel with lot of albumin and so on. A quality measure of a medium-derived 2-D gel is the complete absence of serum albumin!

Ambort


daveytea

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Re: (Dambort)
« Reply #9 on: March 11, 2007, 05:00:00 PM »
Thanks all for the helpful suggestions.
I've been busy with the extraction recently and forgot to check out the thread... sorry for not making any responses previously...

For the culture medium, i guess i will try ultrafiltering the BHI medium to remove all proteins that >10kMW cus it seems that the bacterium cannot grow in other protein-free minimal medium...

For the sample preparation, I have now trying the ultrafiltration method. After concentrating 500ml medium to 10ml, I'm now wondering whether I should try dialysis first before further concentrating the sample using centricon, etc... since the sample looks pretty "dirty" and viscious. I assume that there is a lot of salts and i guess ultrafiltration doesn't involve any step to remove salts. But then I am not so sure what buffer to be used for dialysis. I'm planning to use 10mM TrisCl/1mM EDTA/1mM pmsf. Could anyone tell me if this buffer is suitable?

And for the advice from Dambort, is the washing of concentrates that u've mentioned means dialysis? and what kind of low-salt buffers should be used? Do i need to add protease inhibitors or urea or similar chemicals to prevent the protein from degradation?

Thanks all for the wonderful advices.

Regards

Daveytea


Peter Campbell

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Re: (daveytea)
« Reply #10 on: March 13, 2007, 05:00:00 PM »
Your comment about salts and dialysis suggests you do not understand what ultrafiltration does.  Little things like salt ions go through the holes; big things like proteins don't.  So if you reduce 500ml to 10ml you will have the same salt concentration as you started with but the total amount of salt will be 1/50th.  You should however have retained most of your protein and have increased the protein concentration 50-fold.  You can then redilute your solution with water and reconcentrate by ultrafiltration several times to remove salt.  You can calculate how much the salt concentration should be reduced by the various start and finishing volumes.
Peter.

Ambort

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Re: (Peter Campbell)
« Reply #11 on: March 14, 2007, 05:00:00 PM »
Yes, what Peter Campbell writes is correct. You do not need to do dialysis when you are doing ultrafiltration. Ultrafiltration simply means that you use ultracentrifugal filter units that are subjected to spin forces in order to filtrate your sample (culture medium) through a membrane. The proteins that are smaller than the cut-off value of the membrane simply pass through the membrane. Larger proteins are retained and this fraction is called the retentate (the flow through is called the filtrate). For culture medium you are of course interested in concentrating your protein and at the same time to get rid of salts. This is achieved in one step. The salt concentration of the retentate after filtration is the same as the one of the unconcentrated culture medium, but by washing the concentrate several times with a low salt washing buffer (for example: 20 mM Tris, 1 mM EDTA, 1 mM PMSF) you can deplete the remaining salts of the retentate! Washing exactly means that you make up your final retentate after concentration to the initial starting volume with a buffer and centrifuge again! Three times washing is best to ensure proper desalting of sample! Hope now everything is clear!

best regards

Ambort


daveytea

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(No subject)
« Reply #12 on: March 14, 2007, 05:00:00 PM »
Thanks Peter and Ambort very much for the really helpful comment.

I think I understand the principle of ultrafiltration much better now. I will try the washing steps then. Hope that everything will go fine this time.

Really grateful to all the advices made.

Regards,
Daveytea


HR

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  • Posts: 1
Re: Protein extraction from culture medium
« Reply #13 on: April 13, 2013, 02:02:45 AM »
Hi,

I am also using a complex medium containing yeast extract and peptone. I used a combination sodium deoxycholate-TCA-acetone precipitation. Pellet was brownish and I dissolved it DeStreak rehydration solution. My IEF is just not moving ahead and in spite of extending the clean-up step of 500 V to 4 hours, my maximum voltage is 60 V against a set point voltage of 8000 V. I am using 13 cm 3010 strips.

I have 3 strips and fear the effort is in vain.