Hi, I have been trying to dissolve retina and lens' protein pellets unsuccessfully for the past couple of weeks. Protein was extracted using trisol and everything went well until the dissolving part.
When 1% SDS + 8M Urea was added, the lens' pellet formed a milky solution which will separate once spun at 3500 rpm for 10 minutes. I tried quantifying the supernatant but got horrible yield! So I decided to swirl the tube a bit and let the supernatant mix with the pellet a bit (here I got high yield of protein!). I was told that I shouldn't do that (swirl the tube after spinning)!! But it seemed to work until couple days ago when I used the supernatant for Western blot and had unspecific bands appearing all over the place. Is that the reason I shouldn't have swirled the tube after spinning?
I tried increasing the percentage of SDS to 3% but still couldn't completely dissolve the pellet.
Does anyone know what else I should do to dissolve rat's retina and lens?? I've tried sonicating, I've tried heating them in a waterbath at 50C and tried increasing the percentage of SDS but none of these seemed to work.
Try sonicate short bursts on high intensity (e.g. approximately 3x20sec). We have frequently isolated protein after RNA isolation using Trizol and 8M Urea and SDS should work fine - you just have to adjust the volume you dissolve you pellet in according to the size of your pellet.
If you want RNA and protein from the same sample and that is the reason why you use Trizol I would strongly recommend you to start using Qiagen RNeasy mini-kit instead. The RLT buffer there works fine to isolate both RNA and protein from the same sample. All you need to do is to save ALL flow-through from your washes and leave it @ -20C for 3 days and the protein will precipitate. If you want details on the protocol I'm happy to help - just contact me!
Good luck!
Have you tried more potent denaturaing agents like guanidine hydrochloride and guanidine isothiocyanate? Also, try reducing the amount of protein in a given volume of sample.
I am not familiar with rat's retina and lens but I have encountered a similar problem when I tried isolating protein from mouse brain brain tissue using Trizol. Some proteins dissolve less wel in SDS, it might be a good idea investigating different solvents. The trizol isolation protocol by ambion protocol has some suggestions. In my expeirments I got the best (still not really good though) results using 9.5 M urea and 2% CHAPS to dissolve the protein pellet.
Is there any reason why you are using trizol for isolation? Are you also trying to isolate RNA/DNA from the same tissue? If you are only looking to isolate protein I would suggest trying a different isolation method alltogether. I never managed to get consistent and good protein yields using Trizol. I also noticed that you lose a lot of the smaller proteins in your sample when isolating with Trizol.
Hi, I don't have experience with retina and lens, I found an interesting discussion on research gate about lens homogenization, hope can help you:
https://www.researchgate.net/post/Do_you_have_any_experience_with_the_homogenization_of_rat_lens
Maybe it is the buffer (you said you used trizol and then sds+urea) and/or the method used for homogenization (sonicator, potter, etc).
For retina homogenization read some articles to find out the same things about buffers anf methods
Try to digest in the Papain solution and then dissolve in SDS.
Hope it works
Indeed, maybe you need to add more denaturaing agents and some reducing agents. 2% CHAPS sounds good, you can also add thiourea, DTT or others mentioned above. Probably, in the conditions you've described, the measurment of protein concentration may be incorrect. Maybe you have to reduce the protein amount.
adding thiourea definitely adds in dissolving proteins..you should try ..
Try sonicate short bursts on high intensity (e.g. approximately 3x20sec). We have frequently isolated protein after RNA isolation using Trizol and 8M Urea and SDS should work fine - you just have to adjust the volume you dissolve you pellet in according to the size of your pellet.
If you want RNA and protein from the same sample and that is the reason why you use Trizol I would strongly recommend you to start using Qiagen RNeasy mini-kit instead. The RLT buffer there works fine to isolate both RNA and protein from the same sample. All you need to do is to save ALL flow-through from your washes and leave it @ -20C for 3 days and the protein will precipitate. If you want details on the protocol I'm happy to help - just contact me!
Good luck!
I have had some luck solubilizing proteins with sarkosyl at about 1%. In my case I was overexpressing GST tagged proteins in a bacterial expression vector and had issues with aggregation. This doesn't sound like it is exactly the same as your issue but the sarkosyl idea may still apply.
Thanks everyone for your kind replies.
FINALLY, I managed to dissolve the lens pellets, using 8M Urea (dissolved in Tris HCl pH 8)! Well the pellets were not completely dissolved but I had high yield from the supernatants.
I am going to try to dissolve retina pellets using the same concoction today and see if it will work. Wish me luck!
Dear Elena, what for Tris-HCl concentration did you use to dissolve Urea? How big was your protein pellet and what for final volume of SDS+Urea did you add to your pellet? I've trying as well couple of protocols that did not work at all. I'd be very grateful with your help :) thanks!
Urea nicely disolves proteins. For 2D electrophoresis I use 2M Thiourea additionally. But when proteins are dissolved in such a solution they rather need to be purified before running SDS-PAGE. Maybe I would go in this direction - dissolve them in urea and thiourea, slightly heating - until you dislove them. Then, unfortunatelly purifythem, for example precipitate them and dissolve to have lower concentration in your 1%SDS and 8M Urea. Then count the concentration, but in my opinion, with such concentration of urea your measurment would not be reliable - depends on what method you use.
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