A full and complete explanation about western blotting technique. Watch also SDS-PAGE: • The principle of SDS P... Southern and Northern Blotting Techniques: • The Principle of South...
I havent finished watching this video yet and boy you are doing a great job. I have been looking for someone who explains the protocol and the theory behind it and this is exactly what i need. Please keep posting 👍👍👍😘😘😘😘
The videos on this channel are great and helped me a lot to better understand the techniques. Your teaching method is excellent, and it is very useful to explain the reason for using each substance and solution. I hope you continue this great videos. Thank you so much!
"...from the mouse or from the rat or from the rabbit or whatever!" 😀 Anyway, I'm moving from SDS-PAGE to Western Blotting in my research lab, and this video was very informative, helping me understand the technique and protocol!
How do we determine the precise Time and Voltage required for our protein of interest? Can we look it up somewhere, or is this something that requires some trial and error? Also, if for example, we have 10 different proteins of interest that we are looking for in our samples, does each protein have their own optimal Time and Voltage (I am assuming yes)? If yes, how can we optimize the Time and Voltage so that we get ALL of our 10 proteins of interest on to the membrane without having some of them being left behind in the gel or in the filter paper?
Do you have a control protein blotted as well? I assume your proteins of interest are from a cell lysate, no? A housekeeping protein like GADPH, or something like Actin? Or some bacterial protein if you have expressed the poteins to have them purified later on? If the band size/intensity of the Controls is the same in each condition, then you indeed have expression differences between wild-type and mutant.
OK. So I have questions. 1) Is there a "ladder" for SDS-PAGE? Otherwise I don't see how can I determine specific molecular weight numbers 2) SDS denatures proteins, would that interfere with the primary antibodies from recognizing the protein of interest given that it is denatured? 3) The "sandwich" is placed in PLASTIC container. Why use plastic, a non-conductor, when we are going to electrically push the proteins? Does the transfer buffer gets into the plastic bag, or stay completely outside?
I have a question regarding the secondary AB. if the first Ab is mouse anti-human why isnt the secondary let’s say goat anti-mouse? Isnt it a mistake? Thanks
Short question.: Why do all proteins move to the anode if you prevent SDS-binding with methanol? Whithout SDS-binding some basic/alkaline proteins would be positively charged, wouldnt they?
Hei, proteins here are already bound to SDS, because as I said; before applying the western blotting technique we applied the SDS-PAGE in which all the proteins were bound to SDS. So the Methanol is added to the transfer buffer in only 20%, so during the run, methanol will lightly detach the SDS from the proteins (not completely) in order to enhace their ability to bind to the membrane.
I have a question... I understand that the primary Ab is extracted from something other than a human (such as a mouse or rabbit). I also understand that the secondary antibody is made anti- whatever the species used to make the primary antibody. However, how is the secondary antibody produced? For instance, if you have a human protein, a mouse primary antibody, then would you need to synthesis the secondary antibody in another animal such as a rabbit? What happens if you synthesis the secondary antibody in a human or mouse?
Hello ... The secondary antibody is an IgG that has a specific affinity to the proteins of a certain species such as mouse or rabbit or sheep. It is also produced in an animal by hyper-immunizing (injecting a high concentration of immunoglobulins) the animal with purified immunoglobulin fractions from a normal mouse (or any other animal) serum to produce High-affinity antibodies. Yes, usually the secondary Ab is produced in another animal because you cannot produce anti-mouse secondary Ab in a mouse because if you inject mouse immunoglobulins into a mouse then the mouse will not produce Ab against them. On the other hand, you can produce anti-mouse Ab in humans but usually, we dont produce Ab in humans (ethically). However, if you are studying sheep proteins for example and you use mouse primary Ab you better not use sheep secondary Ab to prevent unspecific binding. So usually for example, if we are studying human proteins we use mouse anti-human primary Ab and rabbit anti-mouse secondary Ab.
I have a preformed a WB and im comparing a wildtype and mutant protein and the results show that we have more intensity in the mutant samples than the wildtype samples. The only thing this tells me is that we have more of the protein of interest in the mutant compared to the wildtype right i.e. it doenst give me any other information?
Do you have a control protein blotted as well? I assume your proteins of interest are from a cell lysate, no? A housekeeping protein like GADPH, or something like Actin? Or some bacterial protein if you have expressed the poteins to have them purified later on? If the band size/intensity of the Controls is the same in each condition, then you indeed have expression differences between wild-type and mutant.
how can i know how much time and voltage i need to my proteins? it depends on what? how can i know if my proteins run away from the membrane or some stayed on the gel?
If you have included your 'protein ladder/standard' in SDS-PAGE (as you should! ;)), then you will see it on your blot (the colours of the ladder are seen)...if not, well, then your samples have migrated into the solution and you can start all over again :p point is, you cannot see it during the run. Make sure you get it right immediately, and stick to your own routine, always, when preparing the cassette! Time and voltage depends. Anyway, you should be careful with higher voltages, as this might increase temperature, and maybe decrease the quality and resolution of your blot. As I remember, we used 25V for 'overnight' (ca. 16h) (and put the whole setup in an ice filled bin!), if you start blotting at the end of the day, OR 45V for intraday (ca. 4h). This worked fine, but ideally you should optimize for your own equipment yourself :)
