Eventhough English is my second language, I’ve not faced any difficulties in understanding your lectures! You simplified and made this lecture and the others easy to understand. I can’t thank you enough
I am a little confused about something: If you see a western blot, containing multiple bands, does that mean that you have created specific antibodies for each of the proteins that make up each band? I mean, in the above example, only one band is visible after the x-ray, the band containing the protein to which the specific antibody would bind. That must mean, that if you have multiple bands that are visible on a western blot, there has been made specific antibodies for each of the bands, right?
it took me so long to understand the single steps and you explained it so easy with everey info i needed. But i have one question: Why not mark the fist Antiboddy with Radioaktiv Isotops? What would be the differen?
I don't think proteins on an SDS polyacylamide gel will move according to charge because the SDS makes all amino acids of the protein to acquire a uniform charge besides protein segregation. so proteins will separate according to size
+Dheer Susania You are kidding right? That isn't even close lol. First of all, it's not a nutcase that want's an award, it a whole different application lady.. and it's not complicating anything. I love when clueless people are like, "hey I know this, let me tell someone how it works".. stop confusing people and read your textbook. WESTERN BLOTTING identifies sizes and shapes of various proteins, incorporating primary and secondary antibodies for identification so that AFTER electrophoresis and transfer to the membrane they can be analyzed. SOUTHERN BLOTTING examines DNA sequences in regards to the detection of specific sequences.. sounds quite different eh? Stop confusing people if you truly don't know what's up. Watch more of the AK lecture's please.
+BWolf 420 Okay you're probably right about the differences between southern and western blotting. But what about the original question about why they can't just radioactively label the primary antibody, instead of having a primary and secondary complex?
I am right about the the difference as I actively use both methods/ analyze their statistical components, which they are much, much different. I think you are misunderstanding the concept of the primary and secondary antibodies. In western blotting the monoclonal antibody binds to specific proteins due to its design features/parameters. All of this happens after electrophoresis. You will have your banding patterns and the desired protein will have the monoclonal antibody attached. It is not yet able to be identified by xray defraction, therefore you design another antibody (secondary antibody) which will be specific to your first designed primary antibody which matches the antigen of interest. It is the antigen-antibody-antibody connection that is needed for valid accuracy as there may be outliers that show up in previous steps. Hope that helps!
hahaha you people are thinking way too complex :D First: Making labelled primary antibodies would technically be possible but it's simply to expensive to label them since making specific targeted AB's is expensive at it is. Second: The secondary antibodies are polyclonal which means they are targeted against every antigen of a specific ORGANSIMS, that includes antibodies made from this organism. Therefore, you produce one batch of secondaries which is able to attach to every monoclonal antibodies derived from the organism e.g. mouse. Happy to help :D
hellow, Mr. Science , i only wanna say that u helped me a lot. because it s unfair to to listen your lecture and not to thank you once. i hope you will introduce us. Molecular marker in lecture coming. thanks for you service to the human
From what I can tell, western blotting protocol is a combination of PAGE and ELISA. Please correct me if I am wrong on that part, but to identify a specific protein can't we just use ELISA instead of western blotting? I probably sound silly asking this, but I am really confused.
the reason is sensitivity and signal amplification ..... if the desired protein conc is very less in western blotting than a few of the primary antibody can bind and giving a less signal ..... so multiple secondary antibody can bind to the primary antibody as a result signal will be amplified and we can detect protein even in the less concentration which increases the sensitivity of the process... hope u liked ,y ans subscribe my channel and post more question and i have lots of videos coming up for u guyzz
I don't know how one person has so much expertise in so many difficult subjects. Sir, you are brilliant!
Eventhough English is my second language, I’ve not faced any difficulties in understanding your lectures!
You simplified and made this lecture and the others easy to understand. I can’t thank you enough
Your notes are fantastic. If you make a review book with your notes from the board I will totally buy it :)
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So clear, thank you for saving me!
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Thank you so much for doing this! I understood it perfectly !
Definitely the best video to explain western bloting
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Great work! Keep striving!
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Exceptional Work as always ! Thank you !
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Thank you! This was very clear and concise!
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leave me speachless every video
I am a little confused about something:
If you see a western blot, containing multiple bands, does that mean that you have created specific antibodies for each of the proteins that make up each band? I mean, in the above example, only one band is visible after the x-ray, the band containing the protein to which the specific antibody would bind. That must mean, that if you have multiple bands that are visible on a western blot, there has been made specific antibodies for each of the bands, right?
very clear lecture, you are the best!
Thanks for all your help, keep up the good work!
you're welcome! will do!
Thanks alot. Your videos are really good.
THIS IS AWESOME! thank you so much!
Thanks for this video..!
Perfect n upto the point💯
You are just amazing ! Thank you so much !
Brilliant explanation!
Clear explanation! thank you!!
it took me so long to understand the single steps and you explained it so easy with everey info i needed. But i have one question: Why not mark the fist Antiboddy with Radioaktiv Isotops? What would be the differen?
Best explanation ever. Thanks man
Brilliant explanation. Change the speed to 1.5x to save time though.
Amazing clarity.
I don't think proteins on an SDS polyacylamide gel will move according to charge because the SDS makes all amino acids of the protein to acquire a uniform charge besides protein segregation. so proteins will separate according to size
Isn't this covered on a 2D electrophoresis gel where it has a pH gradient to differentiate compounds by their charge via isoelectric focusing?
Shouldn’t another protein eg BSA be used to cover all uncovered spaces on the membrane?
He meant they move due to the charge imparted on them in the electric field. He specifies that their position is only dependent on their size.
What's the purpose of using two antibodies? Wouldn't it be simpler to just use a single antibody that is radioactivity labeled?
+Jesse Shooter using single antibody means its southern blotting. probably some nutcase wanted an award, so complicated things to invent a new method
+Dheer Susania You are kidding right? That isn't even close lol. First of all, it's not a nutcase that want's an award, it a whole different application lady.. and it's not complicating anything. I love when clueless people are like, "hey I know this, let me tell someone how it works".. stop confusing people and read your textbook. WESTERN BLOTTING identifies sizes and shapes of various proteins, incorporating primary and secondary antibodies for identification so that AFTER electrophoresis and transfer to the membrane they can be analyzed. SOUTHERN BLOTTING examines DNA sequences in regards to the detection of specific sequences.. sounds quite different eh? Stop confusing people if you truly don't know what's up. Watch more of the AK lecture's please.
+BWolf 420 Okay you're probably right about the differences between southern and western blotting. But what about the original question about why they can't just radioactively label the primary antibody, instead of having a primary and secondary complex?
I am right about the the difference as I actively use both methods/ analyze their statistical components, which they are much, much different. I think you are misunderstanding the concept of the primary and secondary antibodies. In western blotting the monoclonal antibody binds to specific proteins due to its design features/parameters. All of this happens after electrophoresis. You will have your banding patterns and the desired protein will have the monoclonal antibody attached. It is not yet able to be identified by xray defraction, therefore you design another antibody (secondary antibody) which will be specific to your first designed primary antibody which matches the antigen of interest. It is the antigen-antibody-antibody connection that is needed for valid accuracy as there may be outliers that show up in previous steps.
Hope that helps!
hahaha you people are thinking way too complex :D First: Making labelled primary antibodies would technically be possible but it's simply to expensive to label them since making specific targeted AB's is expensive at it is. Second: The secondary antibodies are polyclonal which means they are targeted against every antigen of a specific ORGANSIMS, that includes antibodies made from this organism. Therefore, you produce one batch of secondaries which is able to attach to every monoclonal antibodies derived from the organism e.g. mouse. Happy to help :D
very nicely explained. Thank you :)
Woooow Best video on Western Blotting. teck you man. help me a lot.
hellow, Mr. Science , i only wanna say that u helped me a lot. because it s unfair to to listen your lecture and not to thank you once. i hope you will introduce us. Molecular marker in lecture coming. thanks for you service to the human
Perfect!!
Thank you.
Why can't we directly use radiolabled antibody
From what I can tell, western blotting protocol is a combination of PAGE and ELISA. Please correct me if I am wrong on that part, but to identify a specific protein can't we just use ELISA instead of western blotting? I probably sound silly asking this, but I am really confused.
You are a genius! Godbless you! Well understood
You are absolutely amazing ♥ thank you
what a boss. brilliant explanation
Thank you!
I love you
Thank you
Take the money I give to my University, you deserve it more
What do you do after you isolated the protein in the last step?
But how could you find the concentration of the antigen /POI then after you isolated it?
thank you!
Thank you, sir
What about blocking the membrane to prevent antibodies from binding non-specific to it?
yes thats a good point we have to block the membrane for undesirable binding using a milk that blocks non specific interaction
Thanks for this video
Awesome lecture 👌
Excellent I owe you dude
good job thanks.
You are the best!!!!
thank you so much
Why not just radiolable the primary protein and skip step #5 altogether?
Why do we need a secondary antibody? Why can't we label the first antibody radioactively?
excellent !
Wouldn't denaturing the proteins make the monoclonal aB no longer specific?
thank you good sir
you are a legend
Can anyone explain to me why you cannot visualize a protein using the primary antibody conjugated to a fluorescent molecule?
the reason is sensitivity and signal amplification ..... if the desired protein conc is very less in western blotting than a few of the primary antibody can bind and giving a less signal ..... so multiple secondary antibody can bind to the primary antibody as a result signal will be amplified and we can detect protein even in the less concentration which increases the sensitivity of the process... hope u liked ,y ans subscribe my channel and post more question and i have lots of videos coming up for u guyzz
Thank you sir
How come the primary antibody isn’t radioactive that way we don’t need a secondary antibody?
oh ma gawd this guys amazing
hello, why cant we make the primary antibody radioactive? so no need for the secondary. thank you!
Thank you for the lecture. One question: when should we use ELISA and when should we use Western blot?
You use ELISA first bc it's less expensive and if it turns out positive you check with a western blot which is more expensive but 100% valid
thank you :)
Perfect!!!
More than Excellent
Need more microbiology based videos..
nice one ,,helpful
You should teach my lecturer, 'Mr Giving Out Handouts' lol
love from egypt
Could you Please Add some translation to portuguese
How do we make the monoclonal anti body specifically for our pr that we want to study!?
Jus take ur protein, inject in to mouse , a few days after take out their spleen and u will ur antibody specific for that particular protein
The pandemic only exists on TV, not in any hospital.
Godsend
Thank u angle😂😍
Or subscrite
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I wish i cd see u in real 😍
Thank you!
thank you !