I am preparing cell lysate from control and treated cells with drug. I incubate the cells with RIPA buffer and PI for 60 min on ice then centrifugation for 60 min at 10 000 RPM at 4 degree. Despite the long time of centrifugation, the supernatant is not clear.
Is there anybody faced the same problem? What did he or she do? Is there any explanation?
Depending on the lysis buffer you are using one possibility could be that you release DNA from the nucleus. In particular Triton X100 above 0.7% will do that in many cell lines. Once that happens you will get some gel like stuff which makes it impossible to spin down. Depending on what you want to do next with the lysate another lysis buffer containing a combination of NP40 and Tween 20 worked well for me. Usually a protein lysate should be cleared within 5-10 minutes.
But I expect that the protein is soluble so I will have clear supernatant
Yes, your supernatant should be almost clear after 10K rpm for an hour. Do you have a lot of cells in a small volume of buffer? Also, if you are infecting cells with viruses you may have the virus in the supernatant. Some viruses are big enough to make the supernatant turbid if the titter is high. Another thing to consider is that nature of your drug, is there any possibility your drug is making the liquid turbid?
I agree with Nuria, You may try to warm (40° C) the supernatant for few minutes may the detergents work better. May be to much protein, try to dilute 1:1 the super.
I will suggest you instead of keeping on ice keep your sample on eppi rotter for 30-60 min at 4 degree at 10-12 RPM and then centrifuge it for 12000 g for 7 min then you will not this problem. This step will help in protein solubilization in lysis buffer as it contain detergents.
But keep in mind that your RIPA buffer should have phasphatase and protease inhibitor cocktail.
Good Luck
I have about 500 000 cells/ml and if I will increase the buffer volume I will have less protein. I have a cancer cell line and I think they are immortalized with a virus but I do not think it could produce such high virus. Dhiraj, I am incubating the cells with the lysis buffer for 60 min with vortexing for 15 sec every 15 min I also add PI and phosphatase inhibitor. Roberto, because I did this before and the protein yield was low so I would not dilute 1:1. so do you think that it could be a problem in the centrifuge?
by the way Nuria, the control show the same so it is not a drug problem.
Not only wash the cells first but also make sure your RIPA is clear. I have seen some cloudy substance build up in bottle and the supernatent looked turbid in my lab.
Yes I wash cells two times wuth ice-cold PBS but I will look to RIPA buffer to see if it is turbid or not
Hi. What is the purpose of your cell lysate? Sometimes, depending on the cell type there is some lipids in the sup. Also, is the prot conc is high (I would say more that 2mg/ml) it could be not clear. In any case, I have found no problem for WB with a little turbid cell lysate. Good luck!
I would like to do WB to detect some apoptotic proteins after drug treatment
After I did that foundd that the con. of protein range from 1 mg to 2 mg
but Thanks for all of you for your supporting information
At 500,000 cells/ml, your protein concentration should not be high enough to result in cloudiness. I have noticed this myself with a particular cancer line, but in my case I believe it to be a result of lipids present in the supernatant. You may try switching detergents, and/or increasing the g-force. 60mins is more time then necessary, 13,000xg for 15min works well.
Depending on the lysis buffer you are using one possibility could be that you release DNA from the nucleus. In particular Triton X100 above 0.7% will do that in many cell lines. Once that happens you will get some gel like stuff which makes it impossible to spin down. Depending on what you want to do next with the lysate another lysis buffer containing a combination of NP40 and Tween 20 worked well for me. Usually a protein lysate should be cleared within 5-10 minutes.
Many detergent solutions become cloudy when cooled down. Check your buffer-detergent combination at low temp. Also K+ may influence; K-lauryl sulfate flocculates.
some lipid contents probably be in the supernatant and of course DNA was released from nucleus when using RIPA buffer. I will do sonication and if you do mind the lipid content, you can precipitate your proteins by TCA.
Have you tried clearing it twice? i.e. spin at 10k for 5 minutes then remove to a fresh tube and spin again - The 10k spin for an hour seems excessive (unless it is a large volume), usually protocols call for a 3-5 min 10k spin to clear large organelles.
The solution however might still contain membrane proteins. The detergents may have broken these up some but there are still plenty of fragments e.g. detergent insoluble lipid rafts that will be presen (if you have kept below 4oCt. Membranes are usually removed by a 100,000g spin (usually 30 mins).
What apoptotic proteins are you looking for - if they are nuclear proteins, you may be better switching to a nuclear prep of your proteins to get a clearer answer. Particularly if the proteins are non-specifically localised under normal condition this can skew your control data (particularly if the change is a re-localisation as opposed to an increase in protein expression) making it seem like there is little change.
S
The clarity of the solution could be due to detergent and/or salt coming out of solution especially if chilled . You may also have too little RIPA buffer present to adequately solubilise your cell membrane/protein complexes
It could also be a consequence of not spinning at a high enough speed to pellet insoluble material: as suggested previously ultra-centrifugation at 100,000g is usual BUT if not available then I would suggest spinning at 19k rpm for at least 20-30minutes.
More info on centrifugation conditions is needed. What the 10000 rpms corresponds to x g. The centrifugation force could simply might be not enough.
als
The comments are quite right above: I can clarify the centrifugal force:
We use a JA25.50 Beckman rotor at 19kpm (~43000xg) for 30minutes at 4C.
We generally do it at 16000 rcf for 20 minutes at +4C. It always worked for us. Ensure your reaction mixture or buffers are clear. a slight turbidity is OK. Try to run a gel and check for the bands.
I usually lyse platelets and to remove residual membranes I centrifuge twice at 5000 g for 30' (I have a large volume and I work in bag). Sometime the centrifugation are not enough to have a clear supernatant and in this case I add a filtration step which fit with my protocol. What about your 10.000 rpm to x g? It is essential to know for comparing with other protocols.
Paola
It is probably lipid content of the lysate. Try some of the points given below it may help you
1.If you are planning for western: you can run SDS-PAGE first, if you are not getting smear then its ok
2. Reduce lysis timing in RIPA buffer to 30 min
3. Centrifuge at 14 000 rpm at 4 dc for 15 min and then collect supernatant gently with a 200 ul tip.
NB: Some times this happened to me with different lysis buffer but it did not affect my result significantly.
Good luck
Tapas
I did SDS-PAGE and transferred the protein to nitrocellulose membrane and stained with Bonseau S and I found that there is no problem of having this turbidity
Hi,
May be you can try laemmli buffer. Where you need to load crude lysate.
Tapas
As noted above, g-force - also called RCF [relative centrifugal force], not RPM is the relevant issue. A large centrifuge spinning a large rotor at 10,000 rpm generates more RCF than a small centrifuge with a small rotor. I don't have the formulat at hand, but RCF is proportional to the radius of centrifugation [the distance from the center of the rotor to the bottom of the tube] times the square of the rpm/1000. I am guessing you are using a microfuge, and not a super speed or ultracentrifuge.
I used to do sub-cellular fractionation of eukaryotic cells by differential centrifugation back in the 70s, prepping a whole cell lysate and then spinning it for increasing g-force and time. I used the scheme developed by George Palade. Unlysed cells and nuclei would pellet at 500xg for 5 minutes. Mitochondria took 10 minutes at 10,000xg, lysosomes 20 minutes at 20,000xg, which was as good as could be done in a RC-5B super speed centrifuge. At this point the supernatant was still not clear, containing ribosomes and microsomes. To pellet the microsomes, I had to spin the samples at 100,000xg for an hour. So it's really not a suprize that you were unable to clarify your lysate spinning at only 10,000 rpm - even if you were using a super speed centrifuge.
May be during incubation some cells are getting dead and ruptured leaving there chromosomal material behind which cause of lower density not get separated..which cell line u r using please elaborate the experiment you are running
And how this turbid solution appears...? Its matter a lot on adding some sens-full logic to answers..!
This has nothing to do with released DNA. Release of genomic DNA causes a TRANSPARENT and gelly structure, which is overcome by use of nucleases or sonication. The OP here is asking about a turbid precipitate that appears at the top of the supernatant after centrifugation, along with a precipitate that forms at the bottom of the tube. It's easy to avoid the bottom-precipitate as it's firm. But this top layer of precipitate is almost unavoidable because it easily enters into pipette tip as you suck the supernatant.
If you are working with small amounts of cellular material, it's hard to notice this cloudy white-layer of precipitate that appears after centrifugation of the lysate for clarification. Once you start working with 10 cm dishes or bigger, you notice this. I always get it regardless of the buffer of choice (I've used RIPA, Urea/CHAPS). For western blot, this doesn't really matter. But if you are working with hydrophobic beads like agarose beads for some enrichment purpose, the proteins present in this cloudy precipitate coat the beads via hydrophobic interaction and you get huge random backgrounds appearing in all of your samples, which eventually render the experiment useless. So, for such applications, you HAVE TO get rid of this precipitate.
I found a solution to this. First, I spin down lysate and get rid of the bottom precipitate. Then I take the whole supernatant and filter it through a Durapore PVDF 0.22 um spin-column filter from Merck. You should use a pore size of 0.22 um or 0.1 um. Both work fine. 0.45 um doesn't clear the lysate well!
A colleague suggested that these floating white stuff could be some aggregated proteins in which air was trapped, like hollow protein aggregates. I don't know how much this makes sense but there is no definitive answer to what they could be.
I think it is possibly due to the speed of centrifugation and that you have insoluble particulates present that require higher speeds. It has already been stated that it is a matter of g force not rpm and this is defined by the rotor used (g force can be used to compare any rotor but rpm cannot). Or if using detergent such as TX100 this may be the cause of the cloudiness at low temperatures which I have seen before.
I think is because of genomic DNA release...just soniucate your sample and the transparent gelly-like structure will disappear
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