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.
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!!!!!!
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! 😊
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: ruclips.net/video/On_ZotdZexI/видео.html
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.
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.
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...
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 :)
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!
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.
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
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.
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.
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?
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.
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?
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?
Hello! I need some information about the polyacrylamide gels. Can you please describe me how I can calculate the concentration of acrylamide and the cross-linker? I mean lets say that I need to make a 10% gel. How I find the amount of acrylamide and cross-linker that I need? And what about their percentage (29:1). Am I missunderstanding something? I'm an undergraduate student so excuse my mistakes.
Hello ... if you want to prepare a gel there are many recipes online for polyacrylamide gels do you don't really need to calculate This is one example: (celldivisionlab.com/2013/08/12/sds-page-gel-recipes/) .. But if you want to learn how to calculate then you first need to know the percentage of your stock acrylamide solution (lets say 30%) and then you need to know the final volume of the gel you need to prepare and the concentration of the acrylamide in this gel (normally for one gel 5 ml is enough and for example you want to prepare a 10% gel) the equation is C1.V1=C2.V2 C1 is the concentration of the stock solution V1 is the volume needed of the stock solution C2 is the percentage of the gel V2 is the final volume of the gel Then: 30 . V1 = 10 . 5 --> V1= 1.66 ml is the volume you need to take from the stock solution I hope I answered your question :)
I have a question, if the gel is keep connecting to the voltage and i want to know whether all the proteins may accumulate at the bottom of the gel? Thank you very much
Hello Liu ... thank you for your question ... the proteins do not accumulate at the bottom of the gel ... when they run in the gel they run in different speeds so the small proteins reach the bottom of the gel first, at this point you should normally stop the electrical supply ... if you do not stop the electrical supply the proteins will start running out of the gel to the buffer, in this case you will lose your proteins in the buffer. The proteins will run out of the gel gradually, the small proteins will run out first then the larger proteins will run out when they reach the bottom of the gel, and some proteins are too large so they stick at the top of the gel when they cannot run further.
They do not stop .. you should stop the electrical supply when the proteins reach the bottom .... you can notice that from the blue color (remember I said in the video that the Bromophenol blue is used in the denaturation buffer in order to give the sample a blue color so I can detect it on the gel (5:20 min)) ... so when the blue color is at the bottom you should stop the electrical supply otherwise the proteins will run out the gel ..
+Biomedical and Biological Sciences thank you very much and i clearly understood the concept of sds. i was just confusing about the endpoint of sds and now i know it is similar to the DNA gel.
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?
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.
SDS can not obliterate glycine and this is because glycine does not truly have a R - group (as you know it only has Hydrogen in place of R). However, it is good to be aware that, SDS always obliterate R group of amino acids in a protein so that the charge is always negative regardless of the inherent resultant charge of the protein initially.
Stacking gel has nothing to do with the separation, you need all the proteins to pass through it smoothly and at the same velocity, so you use low concentration (usually 2%) in order to make the gel less condensed with larger pore size. On the other hand, the separating gel should separate the proteins, due to this, you should use a higher concentration in order to make a gel with a certain pore size from which the proteins pass in different velocities (usually 10 to 14%) ...
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.
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?
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
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 best video I found on the internet about SDS PAGE. Thank you so much for all the details.
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.
You're explanation so good
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
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
More detailed than other videos on SDS-PAGE.
yup, stay tuned for more videos :)
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! 😊
I had watched many videos to understand this topic, but this video is definitely the best! Thank you so much!
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:
ruclips.net/video/On_ZotdZexI/видео.html
you have my thanks madam for saving me from mid exam disaster. Thank you so much
I finally undertood Electrophoresis!!!! THANK YOU! Greetings from Argentina ♥
Hei, Glade you liked the video ... stay tuned :)
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.
Really really really good explanation...I was searching for this type of detailed explanation in whole of RUclips.....
This is the most helpful video i have ever watched about sds page. Now i understand much more about this! Thank you so much
For SDS, I just watch this... Thank you for this indepth information mam🙏🙏
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.
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...
Awesomely explained.. Thanks very much mam. Lots of love and respect from Bangladesh
Thank you so much for the explanation, especially for the stacking gel's part! I found it very clear.
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 :)
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.
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 hard work you put into this video! it is very much appreciated :) your video has helped me a lot in the lab today!
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!
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.
you are so amazing, keep going like this!! this is THE ONLY VIDEO i found explained well
I wish I will have a teacher like you in the future. terrific and detailed explanation
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
Very well explained! Do you have any sources for this? I would like to refer to it in my masters.
Thanks Dr. what are the advantages and disadvantages of SDS page compparesmd to the native Page
Thank you so much video is very helpful to understand better SDS page
True
Mam please keep uploading such videos so that we can have clear concept about each technique ur way of teaching is great😄
Why and how proteins have been aligned in the stacking gel? Thank you.
Explanation is fantastic but it will very well if you explain how stacking and separating gel is prepared...
Oh my god so useful video... you save my grade
Very nice explanation of a difficult concept of SDS PAGE 👍👍👍👍
i find it very useful thankyou so much for make that kind of videos
thank you so much, the explanation was very descriptive and reasonably good.
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
This video was very useful. Thank you ma'am
The most helpful video ever! 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 😘🤩
Beautiful explanation! Love this ❤
Congrats! Very helpful video, I understood everything I needed for my lab. Thank you! (the explanation is very good and well constructed)
Thankyou so much for the explanation. I loved it 😊😊. Good work.
Excellent Explanation 👍👏👏👏
Keep it up 👍
Superb video!! Insanely helpful! Thank you so much!!
extremely helpful video for beginners
Thank you very much for the video. You're a good teacher. 😊
Thank you mam .for giving a good visual memory of SDS.page .
This is a really great explanation of d topic
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
Outstanding explanation ❤
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
Very helpful keep it up.
Finally I understood 🤗🤗🤗 thank uuuuuuu
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?
Love the video! Very informative. Thank you so much
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.
Superb explaination
Perfect .👍 simply wht i was actually searching for🙌
Very good explanation. Thank u sooo much mam
thank you very informative ! keep going from Saudi arabia Salam
thanku for clearing my doubts about this techniques
4:27 the disulfide linkage stays intact because its a covalent bond, just like the peptide bond.
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
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?
Very well put together. loved this video Thank you :)
Thnx sooooo much.
Very clear and important
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 :)
Nicely done yet again.
Awesome teaching. Thank you so much.
welcome ... Thank you for the comment :)
thank you mam....for such a well explained lecture.
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?
Very informative. Thank you😍
This was very helpful! Thank you!
Your videos are excellent!
Hello!
I need some information about the polyacrylamide gels. Can you please describe me how I can calculate the concentration of acrylamide and the cross-linker? I mean lets say that I need to make a 10% gel. How I find the amount of acrylamide and cross-linker that I need? And what about their percentage (29:1). Am I missunderstanding something?
I'm an undergraduate student so excuse my mistakes.
Hello ... if you want to prepare a gel there are many recipes online for polyacrylamide gels do you don't really need to calculate This is one example:
(celldivisionlab.com/2013/08/12/sds-page-gel-recipes/) ..
But if you want to learn how to calculate then you first need to know the percentage of your stock acrylamide solution (lets say 30%) and then you need to know the final volume of the gel you need to prepare and the concentration of the acrylamide in this gel (normally for one gel 5 ml is enough and for example you want to prepare a 10% gel) the equation is C1.V1=C2.V2
C1 is the concentration of the stock solution
V1 is the volume needed of the stock solution
C2 is the percentage of the gel
V2 is the final volume of the gel
Then: 30 . V1 = 10 . 5 --> V1= 1.66 ml is the volume you need to take from the stock solution
I hope I answered your question :)
I have a question, if the gel is keep connecting to the voltage and i want to know whether all the proteins may accumulate at the bottom of the gel? Thank you very much
How can they stop if the pores are the same?
Hello Liu ... thank you for your question ... the proteins do not accumulate at the bottom of the gel ... when they run in the gel they run in different speeds so the small proteins reach the bottom of the gel first, at this point you should normally stop the electrical supply ... if you do not stop the electrical supply the proteins will start running out of the gel to the buffer, in this case you will lose your proteins in the buffer. The proteins will run out of the gel gradually, the small proteins will run out first then the larger proteins will run out when they reach the bottom of the gel, and some proteins are too large so they stick at the top of the gel when they cannot run further.
They do not stop .. you should stop the electrical supply when the proteins reach the bottom .... you can notice that from the blue color (remember I said in the video that the Bromophenol blue is used in the denaturation buffer in order to give the sample a blue color so I can detect it on the gel (5:20 min)) ... so when the blue color is at the bottom you should stop the electrical supply otherwise the proteins will run out the gel ..
I hope I answered your question ... stay around there will be many interesting videos on this channel :)
+Biomedical and Biological Sciences thank you very much and i clearly understood the concept of sds. i was just confusing about the endpoint of sds and now i know it is similar to the DNA gel.
Why is it necessary to denature the protein Ist and then separation on the basis of size of protein?
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?
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.
Very good explanation ...really impressed..please make biochemistry molecular biology cell biology related videos to help us..thank you
Does SDS bind to glycine?
is SDS binding to peptide bond or R group?
can you kindly clarify my doubts.
SDS can not obliterate glycine and this is because glycine does not truly have a R - group (as you know it only has Hydrogen in place of R). However, it is good to be aware that, SDS always obliterate R group of amino acids in a protein so that the charge is always negative regardless of the inherent resultant charge of the protein initially.
why do we take different concentration of polyacrylamide gel for stacking gel(5-7%) and resolving gel(12-15%)?
Stacking gel has nothing to do with the separation, you need all the proteins to pass through it smoothly and at the same velocity, so you use low concentration (usually 2%) in order to make the gel less condensed with larger pore size. On the other hand, the separating gel should separate the proteins, due to this, you should use a higher concentration in order to make a gel with a certain pore size from which the proteins pass in different velocities (usually 10 to 14%) ...
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.
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?
So clear ,good job 👍
Thanks for the video. It is very helpful for my bachelor degree project, btw please pray for me.. hahahaha
is the ready made gels have stacking and seperating gels??
What is the mechanism for coloring with coomassie brillant blue colour?
thank you for the video. can you make a video of how to read the protein electrophoresis result?
Can you explain how to prepare stacking and separating gel process
Pls can u answer this question about material that you say (protein )
Why we use half the grain of wheat without an embryo?
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 😊