Dear Nirjhar,

as with all things, the recipes for RIPA lysis buffers vary greatly. We use RIPA buffer for cell lysates to perform Western Blots and our RIPA is very strong and also destroys all membranes of the cells. So I would suggest that it also lyses isolated exosomes of any kind. It is very effective, so we only use small volumes (for example 3x106 macrophages are easily lysed in only 50 µl). I attached for you the formula of our RIPA buffer.

all in H2Obidest:

150 mM NaCl,

50 mM TrisHCl,

1% SDS,

0.5% NP40,

0.1% deoxicolic acid

adjust to pH 7.4

stored at RT

I hope that helps you.

All the best and stay healthy,

Marc

Thanks a lot Mark. My question is specific to exosomes. Systems Bioscience recommends using 200 uL of RIPA for exosome yield from the ExoQuick TC kit. But my question is is there any evidence in literature? How to optimize the lysis of exosomes for protein concentration and Western Blotting? I know that it will varry from cell to cell. But there should be at least a range. I feel that the EV field is often frustratingly disorganized. We should all contribute to standardize every detail in exosome-research.

Dear Nirjhar,

since exosomes are in the end vesicles that enclose cargo with a membrane, the RIPA buffer will just lyse them. So like with any other lysate, you will get protein from your exososomal lysates, which will work with Western Blotting.

Since you do not want to fractionize your membrane from the contents of the exosomes , there is no need for optimization at this point in time. You just want a lysate, which you can use for Western Blot analysis. And for this purpose, the RIPA buffer is the buffer of choice.

The kit states a volume of 200 µl, but you do not know the composition of the RIPA buffer delivered with the kit. So perhaps it is a little bit milder, therefore you need more volume.

Everbody knows how frustrating it can be to establish something completely new, but I would suggest, before you read for months, just give the RIPA buffer a try and see, if you can isolate protein for your Western Blot. If this does not work, then you can search for other methods or start optimizing.

All the best,

Marc

Thanks a lot Marc. You are absolutely right on the general purposes like WB. But the sensitivity of westerns can vary depending on the exosomal protein concentration. Also I need a very precise protein concentration for my experimental design. And yes, as there's no previous reference, I don't have an idea of the typical exosomal protein content in our cells.

You are welcome :) I think a lot of exosomes are necessary to get enough protein for proper WB analysis. Keep me up to date how it worked.

All the best,

Marc

Update on these question from my experience is:

It's not necessary to lyse the EVs or Exosomes with RIPA buffer. The SDS in standard loading buffer for western blot effectively disrupts the exosomal membrane. I routinely do this for melanoma exosomes and Dr. Hector Peinado, a leading EV-expert also told me that he follows this protocol. However, many labs like David Lyden's group lyse exosomes with RIPA prior to loading them on gel.

EV community needs to standardize protocols. Otherwise, existing literature only contribute to more confusion.

I hope this will be helpful for many new researchers or students starting on exosomes. Good luck!

Thanks for your experience, it helped me a lot, Nirjhar

In fact, I wonder if the exosome membrane can be used in other Westernblot after it is destroyed. Almost all the papers I have seen use WB to identify exosomes (e.g. CD63).

I want to use WB or ELISA to detect specific protein in BMSC exosome instead of exosome marker. I don't know if this is possible.

If possible, I just need to replace the CD63 in WB Protocol with other antibodies, is it right?

Thanks

Hi, Nirjhar~ I am confused with the answer of whether RIPA buffer should be used to lyse the exosomes in werstern blot recently, and I m surprised to find the information that you share. Cause you mentioned clearly that "It's not necessary to lyse the EVs or Exosomes with RIPA buffer. The SDS in standard loading buffer for western blot effectively disrupts the exosomal membrane". would you like to share the detail exosomal protocol of western blot? that'll thanks a lot!!!! or could you share relative articles? hat'll thanks a lot, too!!!!

For introduction, i am primierly researching the role of tissue-derived exosomes in cervical cancer, but now was stocked by the characteristic of exosome in western blot. and i searched many literatures that it was vague to mention the necessary of RIPA and exosomes. two days early, i try western bolt in first time, and i did not use RIPA, consequently, the target TSG101 protein marker not shown. the sample i used in western blot is a liver of mice, not cancer tissue.

look forward to you reply~ keke

Hello,

I do agree there is a lot of mixed information available on exosomes and its further use. Adding to the ongoing discussion, I have recently used RIPA buffer for lysing my EVs to be used in western blotting. For me it worked well, though my sample was plasma EVs but I have also seen with breast milk EVs.

I have one more question in this regard, which method you all prefer to go for doing proteomics of exosomes and how to lyse exosomes in that regard?

Any suggestions will be appreciated!!

Dear Nirjhar Aloy

I am working on a similar project and would greatly benefit from your findings. I wonder if you would be willing to share your working protocol for exosome lysis without needing RIPA buffer.

I understand that protocols can be sensitive information, so please let me know if there are any specific details that you would like to keep confidential.

Thank you.

Regards,

Murad.

Nirjhar Aloy just as you've alluded to, our experiments suggest that RIPA disrupts EV membrane harshly compared to other lysing reagents. since the goal is to characterize the cargos for WB, I have then resulted to sonicating the EV in suspension just before loading them on the gel, then SDS or LDS+ME will get the job done afterward.

However to answer your question more specifically on how much volume of RIPA should be used.

1. The starting volume of depleted medium for isolation is crucial- e.g small vol (1-10ml), the RIPA should be the same vol of buffer you want to resuspend the EVs in after precipitation. Say, if you plan to suspend with 100ul of PBs, then split RIPA (50), and PBS (50) totaling 100ul.

2. Once you lyse the exosomes, you can expect that the protein concentration would definitely increase compared to the undisrupted exosomes. So, you might to factor in the appropriate vol for this step.

3. I have compared nonRIPA exosomes with RIPA lysed exosomes by western blot using CD9 antibody and all I can deduce is that the starting volume for isolation, the cell line for culture, and the isolation/precipitation methods are extremely important for exosome characterization. If you are more interested in the isolation mRNA or protein cargo, then there are kits that could do this without totally shredding the membranes as this is also important for some membrane-derived exosome proteins.

Best!

Abayomi Opadele Thanks for sharing your experience. It is no longer a question for me. I have already mentioned my experimental findings and I have already shown that for our cell lines, SDS in the loading buffer is sufficient for optimal protein yield for WB. So, I do not recommend lysis with RIPA as essential. RIPA-lysis and sonication for common EV markers are not necessary in melanoma cells. However, it might be useful in other cells. Thank you.

Hi Nirjhar

Could you kindly tell me how did you measure the concentration of protein? I am using Qubit and getting a very low protein concentration. Do you measure the concentration after lysing them? When you use RIPA buffer, do you use protease inhibitors as well? I am using HT-29 cell lines and starting from a volume of 10 ml. I am going to scale it upto 40 ml. I use PEG to concentrate the EVs and finally do ultracentrifugation when I dissolve the pellets in PBS. Is it the cell number that is causing the problem in my case? Any suggestion will be highly appreciated.

Zubaida

Hi Zubaida,

I always use BCA protein assay. However, Bradford or DC protein assay (biorad) or any other reasonable method should work.

In EV-protein analysis the use of protease inhibitor is optional, most people don't use it. I do not use RIPA. In melanoma cell derived EVs, just the SDS/LDS loading buffer works fine for me.

Do you make your PEG or is it company-made? How big is your pellet?

Hi Nirjhar I make the PEG solution on my own and I do not see the pellet.

Okay, how much conditioned media do you start with?

I start with 10 ml. But I am scaling up to 40 ml. I just isolated some from 40 ml and will.measure the protein concentration tomorrow.

Okay, please let me know if you have any problems. Generally, if your cells are okay then, the issue might be with the reagents. I have isolated and characterized EVs/Exosomes successfully using both ultracentrifugation and kits. Even big shot scientists often acknowledge how difficult EVs are! I'll be glad to help.

Thank you very much for your response. I was finally able to see the bands. However, my membrane does not look very clean, also keeping the EVs for more than 2 weeks in 4°C degraded the protein. Do you have suggestion on how to keep them undegraded for a longer period? Because I use control cell line HT29 and virus infected HT29 cells and isolate EVs from them. I collect the EVs at two different time points, hence one of them has to wait until the other one is ready. I know that EVs go bad pretty quickly. Should I then run them separately in two different gels once each one of them is ready? Also, if you could suggest me some tips on how to keep my membrane clean, it would be very useful.

I can solve one of your issues right now. EVs should always be preserved at -80 degrees C. It is generally stable at -80 for at least a year. Please avoid repeated freeze-thaw cycles.

For the western blot, you need to be more precise, are you having a lot of background or multiple bands? Which markers you are probing for?

Ok so, once I isolate them, I will keep them at -80 C until I am ready to do the WB experiment. I am getting a lot of background but certainly not a lot of bands. I was wondering if I should freshly prepare my running buffer as I keep reusing them. However, the transfer buffer is fresh and I use a semi-dry transfer process. I am probing for CD63.

Hi, it looks like that you still do not have a good yield. A good yield of EVs is generally enriched in tetraspanins. CD63 is a very common marker and I see a very clean, robust band in both ultracentrifugation and Kit extraction technique. People generally do not report the amount of protein loaded in each well. But for CD63, depending on cell type and isolation technique 5-30 ug of EV-protein should produce nice bands.

Zubaida Marufee Islam Background can be short time of blocking or low dilution of 2nd AB. Check recomendations for the brand you're using. Also remember to use a loading buffer without BME so you can see CD3 bands.

Thank you!