I have used this one below with acceptable yields. Please, take in account that the differences in the use of BSA or FBS and/or suplements in your samples may affect your measures of total protein concentration, so may be you will need to adjust based on your blot of your reference protein (actin or other you're using).

- 1 ml (5 ug min. of protein) + 1 ml TCA 20%

- 15-30 min. on ice

- Centrifuge 5 min. at 10000xg and remove supernatant

- Wash in 0.5 ml of cold ethanol-ether (1:1) or acetone

- Centrifuge 5min. at 10000xg

- Remove supernatant, repeat 1-2 times the washing step and let to dry the pellet

- Store at -80ºC or Resuspend in loading buffer 2x at 95ºC by 5 min and proceed to quantification and gel running.

Good luck.

Hi you can use 10 or 12% bis-tris polyacrylamide gel for you rprotein of 30 or 45 kDa. for detailed protocols, plz have look on the refs. Dron et al. 2010 JBC or Salamat et al. 2011 J Virol. If still not clear, plz don't hesitate to ask, i'll be at your disposal for any furtehr help. good luck

Hi, thanks for the answer but what I understood from your publications is that the proteins you were looking were not secreted into the medium. I am looking for a protocol that could allow to do Western Blot on secreted proteins. Do you know a protocol for that?

Cheers

For the protein secreted into medium, you collect the medium and then concentrate the protein by precipitating it with 4 volumes absolute Methanol at -20°C overnight (minimum 2h) (or by TCA etc). In th efirst attempt, you can direclty load your protein on gel by adding equal quantities of the medium and loading buffer.

You can quantify the total protein in the medium before loading on the gel so to pour the equal quantities of different samples.

We did the same with the GPI-deficient prion which was essentially secreted in the outer medium and it worked well. You can see this in the figure 8a and 8b of Salamat et al. 2011 J Virol.

Try this protocol, hope it will work. If still you have any ambiguity, plz don't hesitate in asking. I'll help you to clear otherwise i'll send you the contact of experts working in my ex-lab. They're very good in such stuff and cooperative. Bonne chance :)

Merci!

If your protein is secreted, then the first step of the process will be concentrating your protein. Acetone, salt or even dialysis can be used to obtain a crude protein extract from the medium. Once this is done, you could preferably follow with size exclusion chromatography, to obtain fractions corresponding to the size of the protein of interest. You can quantify those particular fractions using BCA, or UV spectrophotometry, and then load the required amount on an SDS PAGE followed by the Western blot.

I have worked on secreted protein in Media. Please have a look on reference Gupta A. and Chellaiah M.A., 2012 publication

I have used this one below with acceptable yields. Please, take in account that the differences in the use of BSA or FBS and/or suplements in your samples may affect your measures of total protein concentration, so may be you will need to adjust based on your blot of your reference protein (actin or other you're using).

- 1 ml (5 ug min. of protein) + 1 ml TCA 20%

- 15-30 min. on ice

- Centrifuge 5 min. at 10000xg and remove supernatant

- Wash in 0.5 ml of cold ethanol-ether (1:1) or acetone

- Centrifuge 5min. at 10000xg

- Remove supernatant, repeat 1-2 times the washing step and let to dry the pellet

- Store at -80ºC or Resuspend in loading buffer 2x at 95ºC by 5 min and proceed to quantification and gel running.

Good luck.

im writting here for a question actually..If you have done this protocol already, would you give a little more informations about the internal control that you used? Thank you.

Hi Rubina. Unfortunately, I haven't done this experiment. Good luck with yours.

I think the best method is to concenterate the protein first using a suitable filter. Then, you can add your loading buffer and run western blot like normal. Usually, this method  doesnot have internal control (which is a big disadvantage). That is why, for released protein I usually use ELISA method, but it is more expensive.

Good Luck.

I am working on proteins secreted in the medium. To precipitate them I perform TCA 20% extraction. I grow cell with complete DMEM and, prior to TCA extraction, I substitute medium with DMEM not supplemented with FBS.

The protocol suggested by Patricia is almost the same of mine.

So I recommend you TCA extraction if you want a very easy to perform and cost-effective method; after extraction you can add Laemmli or sample buffer and directly load on gel (I suggest 12% polyacrilamide). 

You should consider that results are variable. You could have variable extraction yields and so different WB results.

If you do not eliminate FBS, you will extract all the proteins present in the serum leading to very "dirty" WB signals.

I suggest you to find a constitutive secreted protein (you can find them easily on SPD Secreted Protein Database) of your cell line as positive control; for negative control in literature is largely used GAPDH to exclude that the protein you extract derives from cell lysis, I use RNH1 which is highly abundant in cells but never secreted.

I also want to analyse secreted protein using western blot. Have you tried the TCA and did it work? My question is for doing the TCA how do you ensure you concentrate the same amount of protein. Do you use equal amount of media to do the concentration or you first do the BCA and concentrate equal protein using TCA before doing the western. Please help guys.

I have a question related to it. I also need to do western blot from the secreted protein in the medium, but priorly, the cells will be subjected to treatment with cyokines and polyphenols. So how do I eliminate the treatments given from the media? Precipitation with TCA, etc would also settle the drugs, isn't it? Kindly help

How would you control your sample loading? Tubulin is not a secreted protein so what should I use?

Dear Aditi Gupta, how much condition media you concentrate with amicon filter?

Hi Shirin,

I use the Amicon Ultra-15. Normally centrifuge 13ml of conditioned medium, for 1hr at 4000g (4°C), and obtained ~300ul of concentrated CM.

Hi Ivan, thanks for your response. Hope I get a good result.

I have a related question - for a time course experiment, can you snap freeze the media and store at -80 and do the TCA extraction when all of your time points are harvested?

Patricia Castillo-Briceno

I tried to use TCA for cell culture supernatant. Unfortunately, the precipitated pellet was very hard to dissolve. Could you please let me know how do you overcome this problem?

Thanks

Dhiraj Kumar

I have not directly have had that problem, but got previously some suggestions.

The best is to avoid over drying.

- To dry the pellet: If doing at 95°C, be sure is maximum during 5 minutes; but can also be in the bench (room temperature) by 20 minutes. That depends a lot on your samples.

But if you are trying to rescue a pellet already dried, you can try on helping to resuspend, you can help the process by carefully pipetting with a p10, (taking care of the volume), first to allow the pellet to be detached from the bottom, and after 2 minutes to help the resuspension. Be sure you don´t take sample with you while doing this, preferable if you manage to workout all this steps with the same tip (by sample). Or at least have all the sample subjected to same process of resuspension.

Hope this help.