simply thank you, you are the best teacher in the universe, you make things so easy , your every vedio is amazing!!, thank you for making us so knowledgeable
Omg your lectures are awesome! Ive been watching all the gel electrophoresis videos and its so clear and also in details i dnt have to waste all my time looking for info on the internet anymore. Thank you!!
Thank you for this. You do a much better job at explaining this than my professor. Likewise with a few other videos you have made. I just subscribed and gave your videos a thumbs up. :)
It's a technique of separation and visualization, but not used as a method of purification, as purification implies that the protein is in it's native conformation. SDS denatures all of the proteins present so that they could not retain any activity and lose their binding properties.
wouldn't the SDS apply a strong negative charge on all the proteins and affect horizontal motion towards the negative end in the Isoelectric focusing component? Assuming both SDS-Page and Isoelectric focusing is occurring together.
Im assuming after you run the 2D gel, you can then do a westernblot as you would with a typical SDS gel and then treat with antibodies to determine different post translational modifications of a protein for example?
Question (might be stupid but ok xD) : with the 2D gel electrophorisis can you find out how many polypeptides,from each protein, there are at your first solution? No right? You just separate them based on these two different properties
Thank you very much for your video. I have a quick question, when the pI is added to the SDS page, will the mass and size increase as you’re going down the SDS page?
i have a presentation on isoelectric focusing on tuesday. i face problems in some points i cannot understand immobilized ampholytes, 2D electrophoresis is type of isoelectric focusing capillary method and SDS are also part of it. how to load sample in gel can you explain me
You can´t IP separation must be done first, because it separes based on the IP so you want the protein to be on its natural form and with it´s natural charge (+ or-), once you do SDS you denaturate proteins wich makes them to lose their dimensional conformation, they are long chains and SDS wich is negative binds to them, giving all proteins a negative charge and thats why now you can separate them ut of their size instead due att all of them have the same charge, wich is negative. So IP first then SDS always.
But how do we separate those proteins which may have the possibility of having the same PI value as well as the same molecular mass. I.e they can't be distinguished by 2d technique?? Please advise.
I wonder how you denature proteins after isoelectric focusing to work in SDS-page, if you take gel from first step the proteins are in their isoelectric point and wont move.
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One of the best explanations Ive heard on youtube for any molecular biology technique. Great Job!
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What a clever way to distinguish between different types of proteins
Thank you very much sir 💝
My concept is clear
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This is very helpful for helping me understand material in my molecular biotechnology class.
Wonderful lecture finally understood this
simply thank you, you are the best teacher in the universe, you make things so easy , your every vedio is amazing!!, thank you for making us so knowledgeable
It is just unmatched... Thank you thank you thank you a million
Thank you for this clear teaching. This video helped me for preparing my exam :)
Which exam ?
@@asishswain1259 Genomics and proteomics
@@yaseminacar4329 Are you doing Ph.D in genomics ?
@@asishswain1259 it was my undergraduate exam. I am doing PhD in molecular pharmacology
Omg your lectures are awesome! Ive been watching all the gel electrophoresis videos and its so clear and also in details i dnt have to waste all my time looking for info on the internet anymore. Thank you!!
Thank you, it was a good explanation. Please keep working, there are so many people that need it.
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tammy wong you're welcome Tammy :)
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Thank you for this. You do a much better job at explaining this than my professor. Likewise with a few other videos you have made. I just subscribed and gave your videos a thumbs up. :)
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It's a technique of separation and visualization, but not used as a method of purification, as purification implies that the protein is in it's native conformation. SDS denatures all of the proteins present so that they could not retain any activity and lose their binding properties.
I learned a lot. Thanks!
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very helpful... Thankyou
wouldn't the SDS apply a strong negative charge on all the proteins and affect horizontal motion towards the negative end in the Isoelectric focusing component? Assuming both SDS-Page and Isoelectric focusing is occurring together.
They r done seperately
Nope... they aren't been done together...first you do the isoelectric focusing and then SDS page
Thank you !!
Im assuming after you run the 2D gel, you can then do a westernblot as you would with a typical SDS gel and then treat with antibodies to determine different post translational modifications of a protein for example?
thank you so muchhh!! :D
Yo, thamk you very much mister
Question (might be stupid but ok xD) : with the 2D gel electrophorisis can you find out how many polypeptides,from each protein, there are at your first solution? No right? You just separate them based on these two different properties
Thank you very much for your video. I have a quick question, when the pI is added to the SDS page, will the mass and size increase as you’re going down the SDS page?
Are proteins taken out of the pH gradient gel to apply SDS or is this done inside the gel?
i still dont get it
i have a presentation on isoelectric focusing on tuesday. i face problems in some points i cannot understand immobilized ampholytes, 2D electrophoresis is type of isoelectric focusing capillary method and SDS are also part of it. how to load sample in gel can you explain me
sir plssss continue ur lectures on biochemistry plsss...plssss...plsss.........we r waiting........................
Is this Native PAGE electrophoresis?
Create your own religion! This got to be scientifically religious the way you explain so clearly. Thanks
nice done (Y)
can you reverse the steps and perform mass separation first and PI separation second. Why or why not?
You can´t IP separation must be done first, because it separes based on the IP so you want the protein to be on its natural form and with it´s natural charge (+ or-), once you do SDS you denaturate proteins wich makes them to lose their dimensional conformation, they are long chains and SDS wich is negative binds to them, giving all proteins a negative charge and thats why now you can separate them ut of their size instead due att all of them have the same charge, wich is negative. So IP first then SDS always.
But how do we separate those proteins which may have the possibility of having the same PI value as well as the same molecular mass. I.e they can't be distinguished by 2d technique??
Please advise.
This is to check stream purity. For separation, I would recommend using them in chromatography and looking at affinity chromatography in particular.
I wonder how you denature proteins after isoelectric focusing to work in SDS-page, if you take gel from first step the proteins are in their isoelectric point and wont move.
So those people who disliked this video think they can make a better lol
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