this is why professors should do research and teachers should teach. not always the case; some professors are excellent, lively, dynamic and patient teachers also ...but most just want to get back to overseeing their projects, publishing papers and finishing grant applications before the dreaded deadlines.
This is the first technique video which I have completely understood without any practical experience. Thank you so much for making such a great video.
Hello! This video really saved me! Detailed and exhaustive. I was studying this technique for an exam and didn't understand it really...now it's all clear. Thanks! 😊
OH MY GOD!!!! Thank you SO much!!! I'm a student from Brazil and I was looking for something that would make me understand the electrophoresis tecnique and I was not findind anything so simple and easy like this video! I'm so happy right now... I was almost giving up haha I was reading, and reading, and reading and not understanding at all, but now is everything so clear in my mind because of you...Omg, THANK YOUUUUU!!!!!!
Wow, this is good ... I have a playlist on my channel containing videos about all the electrophoresis techniques, have a look at it, it will help you for your exam: th-cam.com/video/On_ZotdZexI/w-d-xo.html
Thanks for the fantastic video! We just watched your video in class and it definitely helped us understand electrophoresis. I especially loved the pace of your video :)
OMG thanks, my wife has been doing these for like 25 years, now I know what the hell she has been talking about. Now to watch your video on westerns...
I really don't understand why the stacking gel concentrates the proteins. If it is still a gel the proteins will be administered as they are pulled from the solution so they would enter the stacking gel separated. What is stopping the proteins from doing this? And how are the loose glycine molecules related. It doesn't seem like they have anything to do with the proteins, they are just there like chloride.
Thanks so much for a very clear explanation! I tried to understand the technique by reading a description online but it was so confusing, thanks for clearing it up for me!
How can we distinguish the different proteins and their polymorphisms from each other after the separation. If, say our sample is cattle milk and we are interested in casein and all it's various polymorphism alpha, beta, kappa etc. Are we going to use the same or different markers to identify the required proteins.
Umm... I have one question. At the stacking gel, speed of the ions is Cl(-)>protein>glycine(n/-). In my opinion, Cl is faster than glycine these distance gonna be far each other. I wanna know details what is happened in stacking gel and these speed.
I have few questions. 1. Why do we have 8.3 pH in electrodes? What is the significance? 2. Is the gel already made or prepared during the experiment? Thanks in advance.
Hi. at the end of the video, you mention that the proteins are not applied to the stacking gel at the same time, but they do so in the separating gel. What does this statement mean? what is meant by being applied to the gel at the same time?
not clear on how the stacking gel helps. is the principle that in the stacking gel the protein migration speed is restrained by the glycine and clorine sandwiching it either side? so that when the glycine transitions to being more negatively charged and speeds up, it "gets out of the trunk"? need more explanation here.
I had the same question as you, and in case you haven't found your answer yet, I found some helpful information in my lab class's Theory Manual. After the chloride anions form the leading boundary in the stacking gel, the area immediately behind is rich in Tris cations and depleted of anionic species. This attracts anionic protein molecules and causes them to migrate at a rate similar to that of the chloride anions, at least initially, and protein molecules of identical size will be concentrated into a narrow band by the time they reach the separating gel.
@@andrewfoster9823 thx sir, that really helped. however i've two questions remaining: 1) what's the purpose of the glycin when the proteins simply get draged by the Cl- front? 2) how does the glycine behind the sample manage to get past it since it's supposed to stack it before? i know that it speeds up, but that stacking-process sounds as if the glycine fraction wasn`t able to get past it because otherwise there would be no sandwitching. to me, it seems to be more logical if the faster moving thing actually has to be behind the sample in order to stack it.
@x4rrr had the same problem, now I was looking through my script again and came across the answer: Due to the fact, that the chloride-anions migrate at a very high speed through the stacking gel, behind this "layer" forms a zone with a high electrical tension (as @Andrew foster mentioned above, it's effected due to the positive cations of the buffer) The second quickest substance are the proteins, which are migrating through the stacking gel. Little proteins are migrating very fast, into the "positive zone" right behind the chloride anions but can't get much further at this speed, because the negative chloride is restraining them a little bit to do so (negatively charged). On the other side, very big proteins are migrating slowly. To avoid, that they don't get stuck too much, glycin pushes them through the stacking gel, so that's the positive effect of glycin. In Austria we call chloride because of that "leitionen" = leading ions, the glycine "folgeionen" = following ions. In the separation gel then glycin is overtaking the proteins, so they aren't pushed anymore and can separate slowly
They only thing I didn’t get is why do we need the migration buffer (i.e the chloride and Glycine) in the gel if the proteins will be separated from them and travel to the negative side of the gel eventually?
I wanna ask a question it wasn't mentioned in the video What does it mean a monocharged protein? What will be the answer if the question is : Gel electrophoresis separates the monocharged proteins? What are monocharged proteins?
Thank you!! It's comprehensive and concise at the same time. I wonder what would be the technical difference (composition and steps) between this and Urea-PAGE for ssDNA or RNA.
Thank you this helps a lot! the article i'm reading talks about 3 different gels running gel, gradient gel and stacking gel, In this video we are shown separating gel, which one of the two (running or gradient gels) would the separating gel be equivalent to?
can someone explain why the proteins stick at a certain point in the seperating gel? shouldn't they be able to go completely through it given enough time?
Hi, I want to stady the expression of a big protein wich mesure 358kda using western blot. How can I prepare a separating gel with a small gradient to visualize this big protein?
Firstly, Thank you for such nice information. Secondly, Why we need to detect protein based on Molecular weight? can we consider this SDS electrophorosis total protein estimation? proteins can also be detected by spectrophotometer,, so what are the application of SDS electrophorosis and spectronic proteins estimation? Looking forward
ik this is late but after the sds page we get those protein bands separated right? so one band = one specific protein so this protein can be digested and send for identification techniques like mas spec for further studies.
Tris Hcl is used in the buffering system in SDS-PAGE and it is used in different concentrations in the stacking and separating gels to serve in the differences of PH between the two layers.
Thank you for your comments, the buffer used inside the gel, and the migration buffer used in the SDS chamber, which are both water based, are responsible to conduct the current.
Why can't my professor just memorise this video and rap it during the lab. Very useful!
Haha thank you ... maybe you can recommend it to your colleagues ;)
throw that ish on Rap Chat yo, that would be FIRE!
your professor probs done thousands of 'gels' while doing his first research work. I m assuming he's just not interested any more:-))
this is why professors should do research and teachers should teach. not always the case; some professors are excellent, lively, dynamic and patient teachers also ...but most just want to get back to overseeing their projects, publishing papers and finishing grant applications before the dreaded deadlines.
Hehe
This is the best video I found on the internet about SDS PAGE. Thank you so much for all the details.
I just have to say it was the best explantion i've ever heard about anything! Thank you, it answered all of my questions....:)
This is the first technique video which I have completely understood without any practical experience. Thank you so much for making such a great video.
Hello!
This video really saved me!
Detailed and exhaustive.
I was studying this technique for an exam and didn't understand it really...now it's all clear.
Thanks! 😊
You're explanation so good
This is the best video I have seen on the working principle of SDS PAGE. Thank you
thanks for the explanation, the best thing is that you dedicate time to explain the principle of the gel, its the best explanation i´ve seen so far :D
OH MY GOD!!!! Thank you SO much!!! I'm a student from Brazil and I was looking for something that would make me understand the electrophoresis tecnique and I was not findind anything so simple and easy like this video! I'm so happy right now... I was almost giving up haha I was reading, and reading, and reading and not understanding at all, but now is everything so clear in my mind because of you...Omg, THANK YOUUUUU!!!!!!
WOOW .. thank you for the nice comment ... stay tuned, you might find everything you are searching for in this channel :)
This helped me a lot before even doing my practicals. Thank you
Perfect ! I spend time looking for those informtion on internet and you give them (and a lot more) very clearly, good job !
Hello, thank you for your comment ... happy you got what you need from the video :)
Biomedical and Biological Sciences yes i actually have an exam about all those technics soon and you saved my life haha !
Wow, this is good ... I have a playlist on my channel containing videos about all the electrophoresis techniques, have a look at it, it will help you for your exam:
th-cam.com/video/On_ZotdZexI/w-d-xo.html
I had watched many videos to understand this topic, but this video is definitely the best! Thank you so much!
I finally undertood Electrophoresis!!!! THANK YOU! Greetings from Argentina ♥
Hei, Glade you liked the video ... stay tuned :)
Really really really good explanation...I was searching for this type of detailed explanation in whole of TH-cam.....
This is the most helpful video i have ever watched about sds page. Now i understand much more about this! Thank you so much
More detailed than other videos on SDS-PAGE.
yup, stay tuned for more videos :)
Thanks for the fantastic video! We just watched your video in class and it definitely helped us understand electrophoresis. I especially loved the pace of your video :)
OMG thanks, my wife has been doing these for like 25 years, now I know what the hell she has been talking about. Now to watch your video on westerns...
I really don't understand why the stacking gel concentrates the proteins. If it is still a gel the proteins will be administered as they are pulled from the solution so they would enter the stacking gel separated. What is stopping the proteins from doing this? And how are the loose glycine molecules related. It doesn't seem like they have anything to do with the proteins, they are just there like chloride.
I am too pleased to watch your videos, these are very clear concept growing video. Thank you very much keep it up.
Thank you for your comment ... I will ... Stay tuned :)
Thank you so much for the explanation, especially for the stacking gel's part! I found it very clear.
Thanks so much for a very clear explanation! I tried to understand the technique by reading a description online but it was so confusing, thanks for clearing it up for me!
thank you so much for the hard work you put into this video! it is very much appreciated :) your video has helped me a lot in the lab today!
you have my thanks madam for saving me from mid exam disaster. Thank you so much
How can we distinguish the different proteins and their polymorphisms from each other after the separation.
If, say our sample is cattle milk and we are interested in casein and all it's various polymorphism alpha, beta, kappa etc. Are we going to use the same or different markers to identify the required proteins.
Awesomely explained.. Thanks very much mam. Lots of love and respect from Bangladesh
Umm... I have one question. At the stacking gel, speed of the ions is Cl(-)>protein>glycine(n/-). In my opinion, Cl is faster than glycine these distance gonna be far each other. I wanna know details what is happened in stacking gel and these speed.
you are so amazing, keep going like this!! this is THE ONLY VIDEO i found explained well
For SDS, I just watch this... Thank you for this indepth information mam🙏🙏
And somebody has been messing with my brain all this while. Anytime I see you in person, I will give you a bottle of win.
Mam please keep uploading such videos so that we can have clear concept about each technique ur way of teaching is great😄
I wish I will have a teacher like you in the future. terrific and detailed explanation
Very well explained! Do you have any sources for this? I would like to refer to it in my masters.
Very nice explanation of a difficult concept of SDS PAGE 👍👍👍👍
thank you so much, the explanation was very descriptive and reasonably good.
Explanation is fantastic but it will very well if you explain how stacking and separating gel is prepared...
I have few questions.
1. Why do we have 8.3 pH in electrodes? What is the significance?
2. Is the gel already made or prepared during the experiment?
Thanks in advance.
the same to you
Congrats! Very helpful video, I understood everything I needed for my lab. Thank you! (the explanation is very good and well constructed)
Thanks Dr. what are the advantages and disadvantages of SDS page compparesmd to the native Page
i find it very useful thankyou so much for make that kind of videos
Oh my god so useful video... you save my grade
Beautiful explanation! Love this ❤
The most helpful video ever! Thank you so much!
Thank you so much video is very helpful to understand better SDS page
True
Why and how proteins have been aligned in the stacking gel? Thank you.
Superb video!! Insanely helpful! Thank you so much!!
Thank u very much u r wonderful keep making these videos I learnt a lot and many things become clear
Really such a amazing explanation 😘🤩
Hi. at the end of the video, you mention that the proteins are not applied to the stacking gel at the same time, but they do so in the separating gel. What does this statement mean? what is meant by being applied to the gel at the same time?
Excellent Explanation 👍👏👏👏
Keep it up 👍
Thankyou so much for the explanation. I loved it 😊😊. Good work.
This video was very useful. Thank you ma'am
not clear on how the stacking gel helps. is the principle that in the stacking gel the protein migration speed is restrained by the glycine and clorine sandwiching it either side? so that when the glycine transitions to being more negatively charged and speeds up, it "gets out of the trunk"? need more explanation here.
I had the same question as you, and in case you haven't found your answer yet, I found some helpful information in my lab class's Theory Manual. After the chloride anions form the leading boundary in the stacking gel, the area immediately behind is rich in Tris cations and depleted of anionic species. This attracts anionic protein molecules and causes them to migrate at a rate similar to that of the chloride anions, at least initially, and protein molecules of identical size will be concentrated into a narrow band by the time they reach the separating gel.
@@andrewfoster9823 thx sir, that really helped. however i've two questions remaining:
1) what's the purpose of the glycin when the proteins simply get draged by the Cl- front?
2) how does the glycine behind the sample manage to get past it since it's supposed to stack it before?
i know that it speeds up, but that stacking-process sounds as if the glycine fraction wasn`t able to get past it because otherwise there would be no sandwitching.
to me, it seems to be more logical if the faster moving thing actually has to be behind the sample in order to stack it.
@x4rrr had the same problem, now I was looking through my script again and came across the answer: Due to the fact, that the chloride-anions migrate at a very high speed through the stacking gel, behind this "layer" forms a zone with a high electrical tension (as @Andrew foster mentioned above, it's effected due to the positive cations of the buffer) The second quickest substance are the proteins, which are migrating through the stacking gel. Little proteins are migrating very fast, into the "positive zone" right behind the chloride anions but can't get much further at this speed, because the negative chloride is restraining them a little bit to do so (negatively charged).
On the other side, very big proteins are migrating slowly. To avoid, that they don't get stuck too much, glycin pushes them through the stacking gel, so that's the positive effect of glycin. In Austria we call chloride because of that "leitionen" = leading ions, the glycine "folgeionen" = following ions.
In the separation gel then glycin is overtaking the proteins, so they aren't pushed anymore and can separate slowly
@@samuelgantner4609 maybe it helped a little
Thank you very much for the video. You're a good teacher. 😊
extremely helpful video for beginners
This is a really great explanation of d topic
Outstanding explanation ❤
Very well put together. loved this video Thank you :)
Love the video! Very informative. Thank you so much
Perfect .👍 simply wht i was actually searching for🙌
Superb explaination
They only thing I didn’t get is why do we need the migration buffer (i.e the chloride and Glycine) in the gel if the proteins will be separated from them and travel to the negative side of the gel eventually?
Thank you mam .for giving a good visual memory of SDS.page .
Loved the video..
But why we don't use two gels in agarose gel electrophoresis like PAGE
Because agarose is mostly used for purification of DNA/RNA
thank you very informative ! keep going from Saudi arabia Salam
Nicely done yet again.
Pls can u answer this question about material that you say (protein )
Why we use half the grain of wheat without an embryo?
Just amazing....y der are no more videos posted? It will b great if u post about centrifuge
Your videos are excellent!
Finally I understood 🤗🤗🤗 thank uuuuuuu
I wanna ask a question it wasn't mentioned in the video
What does it mean a monocharged protein?
What will be the answer if the question is : Gel electrophoresis separates the monocharged proteins?
What are monocharged proteins?
Very good explanation ...really impressed..please make biochemistry molecular biology cell biology related videos to help us..thank you
Thank you!! It's comprehensive and concise at the same time. I wonder what would be the technical difference (composition and steps) between this and Urea-PAGE for ssDNA or RNA.
This was very helpful! Thank you!
Thnx sooooo much.
Very clear and important
Can you explain how to prepare stacking and separating gel process
Why is it necessary to denature the protein Ist and then separation on the basis of size of protein?
Very good explanation. Thank u sooo much mam
Mam your videos are very good. It is very easy to understand this. Mam can you please make a video on immunoelectrophoresis. Thank you so much 😊
thank you for the video. can you make a video of how to read the protein electrophoresis result?
thanku for clearing my doubts about this techniques
Very helpful keep it up.
Thank you this helps a lot! the article i'm reading talks about 3 different gels running gel, gradient gel and stacking gel, In this video we are shown separating gel, which one of the two (running or gradient gels) would the separating gel be equivalent to?
can someone explain why the proteins stick at a certain point in the seperating gel? shouldn't they be able to go completely through it given enough time?
Hi, I want to stady the expression of a big protein wich mesure 358kda using western blot. How can I prepare a separating gel with a small gradient to visualize this big protein?
Awesome teaching. Thank you so much.
welcome ... Thank you for the comment :)
Firstly, Thank you for such nice information.
Secondly, Why we need to detect protein based on Molecular weight? can we consider this SDS electrophorosis total protein estimation? proteins can also be detected by spectrophotometer,, so what are the application of SDS electrophorosis and spectronic proteins estimation? Looking forward
ik this is late but after the sds page we get those protein bands separated right? so one band = one specific protein so this protein can be digested and send for identification techniques like mas spec for further studies.
Thank you very much!!! I'm just from the lab where i did all this without understanding anything!! hahahahaha
HAHAHA this is normal ... Sometimes we do things without going into the details ... stay tuned in the channel so you can see all the new videos :)
Thanks for this interesting video I have a request what is the reason to use different concentration of tris hcl in staking and separating gel?
Tris Hcl is used in the buffering system in SDS-PAGE and it is used in different concentrations in the stacking and separating gels to serve in the differences of PH between the two layers.
Thank you for your response
Thank you for the really great explanation. I still don't quite understand why the stacking gel is needed in the first place. Can someone please help?
Clear and to the point. Thank you
thank you mam....for such a well explained lecture.
Very informative. Thank you😍
what is the actual charged particle which conduct current in the gel and responsible for movement of charge proteins?
Thank you for your comments, the buffer used inside the gel, and the migration buffer used in the SDS chamber, which are both water based, are responsible to conduct the current.
Do we have to pour 2 different types of gel or the stacking and separating gel form on their own?
Generally it's sold this way u just hv to follow the instruction
What is the mechanism for coloring with coomassie brillant blue colour?
So clear ,good job 👍
This is so useful. Life Saver!
Solid video! Thanks a million times!
Subbed!
very good explanation. Thank u!