Hello, my name is OB Pena. I work in the Michael Car Lab in the physiology and biophysics department of the University of California. And today I will show you to perform a western blot using the new page system from individual gene.
This is a basic technique to analyze expression of protein to check the results of a precipitation experiments. So western blood techniques involve three basic steps, loading and running the gel, transferring the protein from the gel onto a nitrocellulose membrane and immuno probing the membrane. Today I will use this technique to analyze the expression of a protein In translated cells.
So I prepare the sample so the protein sample are always responding in a sample buffer for X.This sample buffer contains dye to help you to load your waste and blood glycerol to help the sample to go in the bottom of the well. It contains also buffer and LDS. This is a detergent negative negatively charge that we could the protein and the protein with some migrate in the electric fields, accounting to the molecular weight and normal to the charge.
You need also to add some reducing agents as DTT to break the ine bone. So the sample are heated 10 minutes at 70 degree to fully the natural the protein and quickly spin on. So now I will prepare the gel.
So we use precast gel, so we need to cut packaging, take the gel, peel off the protection. So now I'm ready to answer the gel in the setup. So when the gel is incorrect orientation, so riding should be facing you and the open parts facing the central part of the cells.
So first you need to close tightly to a leakage and you add some running buffer in the central parts. I know I will add the running buffer in the other parts, the outside parts, you need to put enough buffer to avoid the gel to warm up during the run. No, I will take on take off the C.Yeah, you have to go slowly and vertically so you're loading from the bottom and you're moving up slowly and just be sure to avoid the bubble.
At the end I'm gonna be loading seven samples and I had three molecular weights. This would help me to cut the membrane after the transfer to block different pathways, different antibodies just to know where I can cut the membrane and where are my samples. So here you have the three standards and after transfer will cut in the middle of the standards the membrane.
So here you will have one block bloating with one antibody and here is the seven parts that will be bloating with another antibody. Just make sure when you load the gel that you load all the well and that you load the J the well with almost the same volume to have a nice migration. Now I'm ready to run the J.So we connect the apparatus to the power supply.
So it's only one way to put the lead switch so you can run the gel either at constant voltage or constant current. So we do that constant voltage and for the first part and we just set the voltage to around 60 70 volts. When I apply the current, you will see bubbles that appear that mean that you have a go good conductivity and you will see the proteins starting to migrate to towards the bottom of the gel towards the positive electrode cuts.
Due to the a DS negative chart that code the protein for the first part of the gel, it is called stacking just to stack the protein in one single bonds it's a low IDE concentration and in the second step of the run I will increase the voltage to hundred and 75 volts. It has been one hour and a half. And judging by the position of the standards you, you can know where the your protein is gonna be according to the molecular weight and compared to the molecular weight of the different unborn of the standard that you can see.
So another, the run is over and I will take the gel off of the cells pathogen in the transfer buffer. That is a code using a forceps or spatula. You separate the two parts of the precursion and take, you cut the part with the well I can take the gel carefully and put the gel in the buffer.
No, We will perform the transfer. It's, which means that we will transfer the protein from the gel towards the membrane nitrocellulose membrane. And for this purpose we will use a bi bio rat systems.
It's a liquid transfer. Other system exists like a semi dry transfer depending of what you have in your lab. So the different part of the system are the cube, the transfer system with this things to prepare your sandwiches.
So the principle is the same for phys, it's horizontal electrolysis this time. So you will prepare a sandwich with two pad to maintain the sandwich very wet. Will put your gel your membrane and two wet man paper on each side and put the sandwich in the system and the protein.
Once again, we migrate towards a positive electrode from the gel to the membrane. So you need to add your own transfer buffer in a dish. So you're chasing the bubble out of the pan.
Can you place the first pad at the bottom of the sandwich? Take one whiteman paper, put in the buffer and let's the buffer getting by capillary and plus press whiteman people on top of the pad Once again, get rid of the bourbon. Now you press the gel And your kids were better part of the jail.
So you take your natural membrane, you can either cut an angle or write something on your membrane to remember the orientation. As you can see, I write a number on the top right angle to remember the orientation of my membrane. No, I will we the membrane in the can place the membrane on top of the gel in the good orientation, adding a new Whiteman paper and chasing the bour.
It's the important step in the transfer process is to avoid bubble. You are, it is very important. Do not have bubble between the membrane and the gel, otherwise the protein will not transfer.
You add the last spell And you close this on which, And you insert sandridge in the system. The black part facing the black part of the systems. If you have two gel, you it repeat the same operation with the other one and you fill the trunk with the transfer buffer.
So now I will add the highest block for the transfer, close the system and transfer my protein for one hour at a hundred volts in the code. Can I also transfer your protein overnight at 20 volts? So now it has been waterer and the transfer is over.
So we'll move to the next steps which immuno probing of the membrane. So first thing is to take your membrane out of the sandwich. So you placed the membrane in the, in a cream tree as you can see, know the standards are on the membrane and not on your gel anymore.
To check the transfer efficiency I will perform a staining. It's a reversible protein staining and you runs sustaining with water. So you can see no the proteins standing red and that's transfer is correct.
So now you had the blocking solution, so either PBS or TBS buffer with 5%milk or caffeine or B 2%BSA. It's purpose of that. It's to cover the membrane with proteins and to block all non-specific sites.
So when in the next step you will put your antibody will not non-specifically bind into your membrane. And we leave the membrane like this at least 30 minutes at temperature. After one hour in the blocking solution, the membrane is ready to be probed by the, by the primary antibody.
So you dumped in the sink your blocking solution and you add on your membrane the primary antibody solution. The primary antibody is diluted in the blocking solution also and you incubate your membrane for one or two hours at room temperature or overnight at four degrees Celsius. After the incubation with the primary antibody, you wash your membrane three times 10 minutes with PBS or TBS CONT containing 0.05%between 20.
So if you d your solution in the sink and you add your washing solution or if you want, you can keep your antibody solution at four degree or minus 20 degree. After the three wishes, you incubate your membrane with a ary antibody coupled to the redish peroxidase for one hour at temperature. So in our case we are, we incubated the membrane with a primary antibody directly compared to HRP.
So we, we just make the three rashes and reveal the waste. The Wahe are no hover and the membrane is ready to be revealed. So there is different way to detect your protein on the western blood membrane.
Today we will use a Kim luminescence assay from you take your membrane, you dry it on a wet man paper, you put it in a clean tray or, and you prepare the substrate solution And you apply your solution on your membrane for five minutes. One or two ml are enough for mini gen. Five minute have passed.
No, we're ready to expose the membrane. So you take the membrane, you dry it on the white man paper and you place it in a plastic polish wash or a serum wrap in western blood cassette you drain. So liquid is observing paper you see and you go in the dark room to expose a film on your membrane.
We are in the dark room. No, I'm going to expose a film on the membrane for one minute. This step need to be performed in the dark.
So you will just see the results at the end to a nice one on your film. You can change the exposure time. I'm introducing the film in the developer.
So we Just got our film out of the developer. And to know exactly the size of the band that you can see on your film, you need to position the film on your membrane and to write on your film the position of the protein standards. In order to position the film, you align the, the fluorescent sticker on the marks on the field and you mark the standards.
In this particular experiment, I've transfected htt cells with increasing amount of DNA. Here, here you have the line correspondence on non-infected cells and here, so increasing amount of DNA that correspond to an increasing amount of protein expressed. Here you can also observe non-specific bone.
It's just a background of the antibody because it's also present in nonspecific non-specific cells. After you have phi, you can take your membrane, wash it in PBST. At this point you can keep your membrane and repeat the immuno immunostaining procedure with another antibody.
After stripping your membrane, you can also catch your membrane in different pieces to, to use different antibodies At the same time, you can store your membrane at four degrees. Use in PBST or blocking Solution, I just show you To perform a western blood. As you saw my, my const is expressed in myself.
So I wish you the same luck with your Experiments.
