This is the most basic salting out and dialysis method used in protein purification experiments. If you need a video explained in Korean, you can see it at the address below. • Salting out and Dialys...
thanks for the Vedio , I have a question it will be great if you could answer that, so after replacing the dialysate buffer what you have added to perform dialysis?
Thank you for your question. You can think of it as constantly replacing the dialysis buffer with a new one for dialysis. If our answer is insufficient, please let me know again. I wish you all the best in your research.
안녕하세요~ 영상 봐주셔서 감사드립니다. 본 영상은 시연용으로 제작되었는데, 저희 실험실은 salting out 하려는 대상 sample에 따라 activation buffer를 적절하게 맞춰 사용하기도 합니다. 그러나 증류수를 사용하여 최적의 온도에서 activation 시키실 경우 큰 문제없이 사용 가능하십니다. 혹시라도 추가로 궁금하신 점이 있으시면 언제라도 말씀해 주시면 감사하겠습니다. 수행하시는 연구 모두 좋은 결과 나오시기를 기원드립니다. 항상 건강하세요.
Hello, thank you so much for making this video, it really helped me. I want to ask something. After the dialysis, I use BaCl2 and HCl to check if the dialysis process has already been completed or not. But there's always a white precipitate and after I try to shake, the precipitate is gone. So is it alright if i stop the process? Thank you
Thanks for the question. There are many factors that occur in what you said, and I do not know under what conditions the experiment was conducted, so I will tell you from my experience. The most common cause: There is a problem with the dialysis membrane itself or a problem occurs during the swelling process. Also, if the dialysis buffer is replaced every 2 hours and the dialysis time is extended longer, the problem has been solved. In a very rare situation, when the protein extraction buffer is not compatible with the dialysis buffer in the next step, a different extraction buffer or dialysis buffer is used to solve it. I wish you good luck with your research.
Thank you for watching. You can use our videos in your school projects as long as the source is open. We hope to advance research in the field of biotechnology through the exchange of various information among many people. thank you.
Hello.. Thankyou for this video.. But i have several questions.. It would be very helpful for me if you could answer them all 1. I want to perform salting out by adding NaCl to a final concentration of 0.9M.. But i didnt understand the meaning of final concentration and should i use the salt directly or i have to make a solution of NaCl first? If yes then what molarity should i use 2. Also for how long i need to add salt.. Like how will i know that saturation is reached? 3. I need to perform dialysis against 0.1 M acetic acid after salting out so my dialysate buffer is 0.1M actetic acid in which i had to keep my dialysis membrane to activate it just like you did?
Thank you for your question. Let me explain based on my understanding of the question. 1. We are going to salt out NaCl to a final concentration of 0.9M. However, without understanding the meaning of final concentration, should I use salt directly or make a NaCl solution first? So what molarity should you use? -> It is thought that the final 0.9M NaCl is added to the protein solution to salt out the protein. Therefore, considering the volume of the protein solution and the molecular weight of NaCl, it would be best to add NaCl powder to the protein solution by mixing it well with a magnetic stirr. If you add NaCl solution to the protein solution, make the same volume of 1.8M NaCl solution as the protein solution and mix 50:50 to make the final 0.9M. 2. And how long should I add the salt? How do I know when saturation has been reached? -> It is recommended that salt in powder form be added slowly. There is no specific time, but in our case, we add additional powder after confirming that the small amount of powder has been properly dissolved. However, protein denaturation occurs when heat is generated, so it is recommended to be careful not to increase the temperature. 3. Since I need to perform dialysis against 0.1M acetic acid after salting out, should I activate the dialysis membrane so that the dialysate buffer is 0.1M acetic acid? -> There is a method recommended by the manufacturer to activate the dialysis membrane. We recommend that you follow it. If you cannot confirm, please contact your dialysis membrane manufacturer by e-mail and they will be able to guide you on the best activation method. When we mainly dialyze protein solutions, we activate the dialysis membrane with distilled water because we follow the method recommended by our dialysis membrane manufacturer. I hope you achieve good results in all your experiments.
Thankyou so much for your reply.. I am having 150 ml of solution in which I need to add salt to salt out protein of interest.. So i read somewhere that if I calculate the grams of NaCl require to salt out protein by taking the volume as 150 ml (bcoz this is the volume in which I need to add salt) therefore 0.9M × 150ml × 58.44 divided by 1000 which comes out to be 7.89 grams. Therefore I need to add 7.89g of salt little by little to my solution. Is that right?
@@aimanalvi9159 I'm sorry for late reply. I was late in checking comments due to the seminar. Looking at the information you wrote, if you add 7.89g NaCl to 150ml solution, it becomes final 0.9M. thank you :-)
안녕하세요~ 질문 감사드립니다. 실온 또는 4도 조건에서 투석을 하시면 되십니다. 그렇지만 초반에는 투석시 교체되는 salt의 양이 많기 때문에 실온에서 버퍼를 자주 교체해 주고 어느정도 salt교체가 이뤄지면, 장시간 투석을 하는것이 dialysis의 효율을 높이므로 단백질 변성 및 미생물 생장 등을 방지하기 위해 4도 조건에서 해주는 방법을 사용합니다. 수행하시는 연구에서 모두 좋은 결과 나오시기를 기원드립니다.
