Hi,

do you experience any insoluble protein remnant when you prepare our sample for SDS-PAGE? Just because air drying over night appears to be very long, usually if pellets are too dry its difficult to dissolve them, usually the protein pellet should dry within 5-10min after Acetone precipitation.

hello, as I can see your protocol is fine so I would suggest to you to try to do precipitation with methanol 100% instead of TCA, or you can use TCA too but if you use it with methanol you will reduce the amount of TCA. Also, with methanol you require less time for the precipitation, around 10 minutes. if still you don't have good data, you need to check your cells whether they are in good conditions to produce enough amount of protein for your precipitation because this is fundamental.

Sebastian Hoernstein

hi! I did not see any insoluble precipitation after I added the loading buffer. Maybe overnight is too long, I will shorten the time and have a try.

Did acetone not dry immediately affect the results? I remember one thing and I am not sure whether it is important that after removing the acetone, I close the cup of 1.5ml tubes and bring it to the other place(cause my lab is in another school). After about one hour, I arrived at my lab, then I continued to dry the sample.

Iskander Madhi

Hi, I agree that protein quality is a key point. Because I tried to do the western blot and detected my protein successfully. Maybe I should improve this in advance.

Also, thank you for your suggestion about precipitation with methanol; I will have a try!

What was the TCA concentration in percentage for your precipitation solution?... It is critical to adjust the accurate percentage in the final recipe to induce precipitation.

Overnight incubation at 4 C followed by acetone wash works well if the sample has sufficient protein concentration to be precipitated. Thus, the second question is what was the concentration you found using protein assays in your sample before precipitation?

İsmail Emir Akyildiz

hi, the final concentration of TCA is about 9%.

4C is also a good idea, thanks!

I often use about 1uM protein, and I can always see it clearly in the gel before I use it to do the assay.

In my experiments, I typically follow the procedure like;

TCA is added to the extract to a final concentration of 10 to 20% and the proteins are allowed to precipitate at 4C overnight (1:1, v/v, sample to TCA solution)...Next, three replicates of ice-cold acetone wash are applied... afterward, the dried protein pellet is dissolved and the protein amount is calculated using BCA to see the best TCA final concentration in terms of protein recovery (precipitation efficacy)...

Good luck