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
+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
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?
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 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
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
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
You should make posters of your white boards
This guy is awesome ☺️
Your notes are fantastic. If you make a review book with your notes from the board I will totally buy it :)
Your explanations are just AWESOME! Congratulations and thank you very much!
thank you so much you have no idea how much you've helped me. all your videos are great!
Great courses! I've learned them for "just a moment" :D
this guy has a unique ability which makes every topic become easier to understand
Sir, you are a G! I will literally pass my subjects because of you. May you be blessed
Again this channel saves me. My text was so confusing but this really straightened it out for me
you explained everything what my professor taught in 2 hours!!
So clear, thank you for saving me!
love your lectures! thank you!
Thank you so much for doing this! I understood it perfectly !
Great work! Keep striving!
Thanks bro, you explain better than all of my professors combined.
Seriously your videos are like the only TH-cam lectures I even watch anymore lol
Exceptional Work as always ! Thank you !
Definitely the best video to explain western bloting
Thank you! This was very clear and concise!
You are absolutely amazing, thank you!
THIS IS AWESOME! thank you so much!
i call u the 'operation sir' the energy u exert is really contagious , best conyer, communicater,
bro this is so clear, i understand it immediately. thanks!
What do you do after you isolated the protein in the last step?
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
Thank you for this, helped me a lot!
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?
Thanks for all your help, keep up the good work!
you're welcome! will do!
Very informative ! thanks for the efforts
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?
very clear lecture, you are the best!
Clear explanation! thank you!!
Thanks alot. Your videos are really good.
But how could you find the concentration of the antigen /POI then after you isolated it?
Thanks for this video..!
Perfect n upto the point💯
You are just amazing ! Thank you so much !
leave me speachless every video
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
Best explanation ever. Thanks man
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.
Brilliant explanation!
Perfect!!
Thank you.
very nicely explained. Thank you :)
Amazing clarity.
Why can't we directly use radiolabled antibody
You are a genius! Godbless you! Well understood
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
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.
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
Woooow Best video on Western Blotting. teck you man. help me a lot.
Wouldn't denaturing the proteins make the monoclonal aB no longer specific?
hello, why cant we make the primary antibody radioactive? so no need for the secondary. thank you!
You should teach my lecturer, 'Mr Giving Out Handouts' lol
You are absolutely amazing ♥ thank you
what a boss. brilliant explanation
Brilliant explanation. Change the speed to 1.5x to save time though.
Excellent I owe you dude
Thanks for this video
Why do we need a secondary antibody? Why can't we label the first antibody radioactively?
I love you
Thank you
Take the money I give to my University, you deserve it more
Awesome lecture 👌
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
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, sir
How come the primary antibody isn’t radioactive that way we don’t need a secondary antibody?
thank you!
You are the best!!!!
you are a legend
thank you so much
good job thanks.
Why not just radiolable the primary protein and skip step #5 altogether?
oh ma gawd this guys amazing
excellent !
thank you :)
Perfect!!!
Thank you sir
nice one ,,helpful
thank you good sir
Need more microbiology based videos..
Could you Please Add some translation to portuguese
More than Excellent
love from egypt
The pandemic only exists on TV, not in any hospital.
Thank u angle😂😍
I LOVE YOU
Godsend
I wish i cd see u in real 😍
Or subscrite
Thank you!
thank you !