+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
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?
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
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?
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
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.
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
