I am working on the secreted active form IL-1beta currently. I used chloroform and methanol precipitation. Briefly, add 1/4 V of chloroform and 1 V of methanol to the harvested medium. Spin at RT/12000 rpm for 5 min. Aspirated the methanol layer and add 1 V of methanol again. Spin at RT/12000 rpm for 5 min again. You can see the nice pallet at the bottom of tube. Air dry or dry on heat block 50 degree C for 5 min. The dry samples are ready to subjected to Western analysis. Another issue I have been struggle was the specificity of antibody. I have been tried more than 6 commercial antibodies and finally found antibody from R&D works nicely. Here is the cat # AF-201-NA. Gel suggestion: 4-12 % NuPAGE gel run on MES buffer system. It separated low molecular proteins nicely. Good luck!

Perform ELISA for IL-1 beta. No need for precipitation

ELISA is the best option, however, if you still want to precipitate proteins from the supernatant, I have used these protocols and in my hands, have worked perfectly. http://wolfson.huji.ac.il/purification/Protocols/ProteinPrecipitation.html

Good luck

Unfortunately I have not tested any protein precipitation protocols for this purpose.

I use Vivaspin concentration tubes of MW cut-off 5 kDa to concentrate IL-1beta from THP-1 macrophage culture supernatants for WB. I start from 5 ml media (from a 25cm2 culture flask of macrophages) and concentrate down to ~100-150 ul, and by loading 1/4 vol of the sample, I get very strong signals for both 17 kDa mature IL-1b and the proform. Could it be that you have just too low amount of starting material for the precipitation?

You can try TCA precipitation, though I would suggest to do ELISA for IL-1beta as it is straight forward and quantitative.

If you are running western blot, the antibody you are using may also be a problem. Several of the ones from Cell signaling work. I can detect p17 without precipitation if the concentration in the supernatant is over ~300pg/ml by loading 40ul on the SDS gel and using super signal femto substrate to develop the blot. If it is less than 300pg/ml, you will need to concentrate or precipitate the supernatant.

I agree with the answers above, an ELISA for IL-1beta would be an easy and straight forward method to quantify IL-1beta concentration in your supernatant.

thanks for your reply but reviewer asked for WB so I have to do the percipitation so I tried the acetone choloroform but I did not get the active form. If you can give me nice percipiotation protocol to get small protein would be great

Once I tried TCA/deoxycholate precipitation, but it didn't work very well for me and it doesn't allow direct MS analysis. You'd have to run the protein in a gel first, but if you want to do a WB is fine.

Alternatively, you can find an overwiew of different protein precipitation/enrichment methods in Chevallet et al, Proteomics. 2007 June ; 7(11): 1757–1770>

I used to concentrate secretomes with a Millipore concentrator filter with a 3 kDa cutoff. It took ages, but it worked and din't require anything more than a tabletop centrifuge. The cutoff is lightly higher than 3 kDa, however. I have not seen anything smaller than 7-9 kDa in my 2D-electrophoresis, so be careful.

The very old method can work to precipitate it without loosing its activity is salting-out with ammonium sulphate (AS). Then you will rehydrate your protein, will not be denatured like in case of TCA or chloroform where you are loosing activity. I use for the small volumes saturated one AS (app. 70%) adding to 100ul of the sample, 50ul of AS, 5 min on ice, then keep the pellet, and another 50-80 ul of AC. There are almost no proteins left in the sup. when AS is 50%. Also daes not make sence to test more, than four AS conc. All the centrifugation you perform at 4deg in any microcentrifuge, top speed, 10-20min. Once you will set up which conc. is working you will have an reproducible method. Rehydratation you perform in any buffer for your reaction.

If your cell culture system allows use Opti-MEM which has very low serum (2%). Thus, your protein concentration is considerably low to start with. Second, you could use molecular cut off tubes to further concentrate. Please remember that Western blot sensitivity is in the range of microgram whereas ELISA can detect at picogram level.

Thus, ELISA is the best choice for the time being.

Carefull with the ELISAs. Most of them cannot distinguish pro-IL-1b from mat-IL-1b! TCA or acetone / ethanol precipitation worked for me. cut-off columns did not work out for me so far. If you use protein precipitation, be aware that you need to use medium without FCS or BSA since this will also precipitate and then loading a gel will be a problem.

There are different mechanisms that fascilitate release of pro-IL-1b or mat-IL-1b, respectively. This might be of interest to you:

Pelegrin, P., Barroso-Gutierrez, C. & Surprenant, A. P2X7 receptor differentially couples to distinct release pathways for IL-1beta in mouse macrophage. J Immunol 180, 7147–7157 (2008).

As Elma said the reviewers asked for WB, so I suggest to skip the discussion about ELISA since there is not to much time left to re-send the manuscript.

The opinion of Krishnaswamy Kannan "Please remember that Western blot sensitivity is in the range of microgram whereas ELISA can detect at picogram level" is wrong! I have found on two pages of the known ECL producers visible WB bands, where protein was loaded at picogram amount. This WB high sensitivity is in agreement with my experience. Microgram level detection on SDS-PAGE is possible to reach only with the worst Coomassie protocol I can imagine.

Dear Elma, if the ammonium sulphate is not working, please try the PEG 4000-6000 precipitation is milder (higher chance to have active protein later).

Checking that your antibody picks up all forms is a good start as suggested previously. Ensure the band does not run off your gel. I do not know what conditions you are using but the use of gradient gel is not the best in this case. Use a high concentration gel and prevent overtransfer.

I used 15% gels to visualize mat-IL-1b. This is sufficient to separate a 10 kDa marker from the running front, thus enough room left for 17 kDa mat-IL-1b.

Actually i have activated human macrophages using LPS and ATP (As I am working on inflammasome). I did the TCA and ammunium sulfate and I run SDS-PAGE today finger cross for me!!!!

First, your cultures have to be serum free, it works well with human or mouse cells. Second, the mature IL-1b (17kDa) will be form under priming of cells with LPS or IFNg for 3 hours. If you don't prime, you will observe just pro-IL-1b. Third, a regular precipitation with methanol/chloroform works perfect. Remember that by ELISA you won't answer the question about mature IL-1b.

Exactly, therefore I prime with LPS and then I use ATP. Unfortunately the methanol/chloroform did not work, but tomorrow my WB would be ready usiing TCA and I used 16% gel. I hope it works. Anyway thanks

I thought that you always get the cleaved IL1beta, but, it was just not detected by specific antibody(Please check whether the Ab is detected only full form of IL1beta) or during the prep, your protease inhibitor wasnot proper.

that method can fish all forms of IL1beta.

Good Luck!!

You can try TCA precipitate.

1. The media was spun at 13K for 2min to get rid of any dead cells and then precipitated with 15% Trichloroacetic Acid (TCA) on ice for 1-2 hrs.

2. The precipitated proteins were pelleted by centrifugation at 14000g for 30min.

3. The pellet was washed once with 100% ethanol and then resuspended in 1x sample buffer and boiled.

I am working on the secreted active form IL-1beta currently. I used chloroform and methanol precipitation. Briefly, add 1/4 V of chloroform and 1 V of methanol to the harvested medium. Spin at RT/12000 rpm for 5 min. Aspirated the methanol layer and add 1 V of methanol again. Spin at RT/12000 rpm for 5 min again. You can see the nice pallet at the bottom of tube. Air dry or dry on heat block 50 degree C for 5 min. The dry samples are ready to subjected to Western analysis. Another issue I have been struggle was the specificity of antibody. I have been tried more than 6 commercial antibodies and finally found antibody from R&D works nicely. Here is the cat # AF-201-NA. Gel suggestion: 4-12 % NuPAGE gel run on MES buffer system. It separated low molecular proteins nicely. Good luck!

For posterity's sake (this is an old question...) I think it's relevant to mention that the cleaved form of IL1b can be blown through a normal PVDF membrane if not handled carefully (it's a small MW). It's best to use a 200 nm pore size Nitro membrane. 1 hour at 100V works well.

Otherwise, the standard TCA protocols have been good for me, and are much easier/safer than methanol-chloroform. NMWC filters are expensive, so I try to avoid them where possible. Also, it helps greatly to do as much of the secretion stage as you can in serum-free medium.

 To Kristiina Rajamäki

I have tried a lot to detect the active IL-1beta in the BV2 microglia supernatant and tissue lysate as well. But could not be able to detect the active Il1beta. Can you provide me the antibody cat and detailed  protocol.

Thanks

I frequently use standard tca protocol to check pre and mature il1 beta in culture supernatant. 

Ajay,

Probably the best antibody for detection of murine IL-1b is AF-401-NA (sample size as AF-401-SP) from R&D Systems: https://www.rndsystems.com/products/mouse-il-1beta-il-1f2-antibody_af-401-na

I use it at the following dilutions:

Western Blot: 1:1000 (IR detection)

Immunofluorescence: 1:200

I have also had very consistent and high quality results with the mouse IL-1b ELISA kits from R&D systems, but I believe there are a couple of good ones from Biolegend as well: https://www.rndsystems.com/products/mouse-il-1-beta-il-1f2-duoset-elisa-15-plate_dy401

Thanks Jordan and Erivan I will try with this antibody.