Speaking of Western Blotting , it is estimated that all technicians will think of black strips on the blue background, or black and white pictures scanned by the instrument. After a long experimental process, the results will be clear, in line with the perfect results of experimental theory, or more of the other endings that make you slightly disappointed.
Compared to other molecular biology experiments, the Western Blotting experiment seems to be more lengthy and more challenging. From the preparation of the experiment to the final exposure, the negligence of any step may be a thousand miles.
Here, BioTNT I put down the complicated and complicated steps, and talked with you about the basics of the Western Blotting experiment.
Don't underestimate the little thing about making glue! The Western experiment started from electrophoresis (the previous sample processing was calculated in the experimental preparation), so the quality of the electrophoresis gel directly affected the final experimental results, in which the quality of the polyacrylamide gel played a decisive role. .
What happens if there is a flaw in the production of glue?
It’s hard to match the good separation glue, only half of it is solidified! Can't use it, can only be remade! Then you must be a newbie, and what is leaking is the easiest to appear.
At the end of the exposure, it was found that the strips were not on the same horizontal line. The strips of the same kDa value should be up and down like a wavy line. Well, the glue should be uneven during the preparation. Tragedy, start from scratch!
Still at the end of the exposure, it was found that there was a small strip in the vertical direction (or black spotting), which unfortunately affected the target strip. Well, that's when you mix a little bubble into the glue, and the proteins are detoured when they run the glue!
Continue, the end of the exposure, found that the destination strips and non-specific strips huddled together, difficult to divide, can not be used as data, this situation is more complicated, may be due to insufficient electrophoresis time, poor antibody quality, in the sample The protein is overdose, etc. However, have you ever thought about it, maybe you have chosen the wrong concentration of glue?
There are also problems such as non-coagulation of glue, uneven size of exposed strips, bad glue when pulling combs, sample overflow during sample electrophoresis, etc., and may have problems with the small details of your glue!
How about it, make a small amount of glue, and eat a big loss! Still look at the problem of making glue well and seriously!
Speaking of this glue, many teenagers still don't know what glue is made (ah, I said, it is polyacrylamide gel!)? In molecular biology experiments, there are two commonly used electrophoresis gels: agarose gel and polyacrylamide gel. What is the difference between these two gels? Keke, the essential difference! Agarose gel electrophoresis is an electrophoresis method using agarose as a supporting medium. The main difference between the analysis principle and other support electrophoresis is that it has the dual functions of “molecular sieve†and “electrophoresisâ€. Its pore size is quite large and is now widely used in the research of nucleic acids. That is to say, the agarose gel can distinguish between the amount of charge and the substance of large molecular weight.
Focus on our polyacrylamide gel, which is a polyacrylamide polymerized cross-linked gel with a network structure and is used as a support for electrophoresis. Prior to electrophoresis, the protein is depolymerized by adding a reducing agent ( opening the disulfide bond of the protein ) and excess SDS (anionic detergent) to the sample , and binding to the protein to form a strongly negatively charged complex to mask the protein. The difference in the original charge makes the charge/mass ratios of the various proteins the same, so the mobility during electrophoresis in a polyacrylamide gel depends mainly on the size of the protein molecule. That is to say, a polyacrylamide gel can distinguish substances having a relatively small molecular weight.
So, what are the ingredients in the polyacrylamide gel?
The first thing to note is that the polyacrylamide gel in electrophoresis is actually composed of two (or more) polyacrylamide gels of different concentrations. They are called concentrated gel and separating gel, respectively. The concentrated gel is 4 % polyacrylamide gel, the main role is to reduce the height difference of the sample when loading, so that all proteins can be on the same starting line, and then get more beautiful and more scientific data. In some laboratories, no concentrated glue is used. This is not worth promoting! The separation gel is a true molecular sieve, and the proteins of different molecular weights are separated by a stereoscopic channel formed by polyacrylamide. In some laboratories, the separating glue is made of gradient glue, which is to use different concentrations of glue to stack in turn, the obtained glue separation effect is better, but the steps of making rubber are more, and the possibility of failure is greatly increased! It is never recommended for novices!
The components in the separation gel and the concentrate are basically the same and are composed of ultrapure water, polyacrylamide, Tris hydrochloric acid, SDS , AP , TEMED . Let's talk about their role!
Ultrapure water: ...not to say
Polyacrylamide: It is prepared by mixing acrylamide ( abbreviated as Acr) and cross-linking agent N , N '-methylenebisacrylamide ( abbreviated as Bis) in a certain proportion, and molecular sieve to separate proteins of different molecular weights. By the way, acrylamide is a neurotic poison! Be especially careful when operating! Polyacrylamide is relatively toxic, but pay attention!
Tris Hydrochloric Acid: Buffer to ensure the pH of the gel is normal.
SDS : sodium lauryl sulfate, an anionic detergent, breaks the hydrogen bond and hydrophobic bond of the protein, and combines with the protein molecule to form a complex at a certain ratio, so that the amount of negative charge of the protein far exceeds its original Some of the electric charge masks the natural difference in charge between various protein molecules.
AP : ammonium persulfate, a catalyst that accelerates the formation of a stereostructure of polyacrylamide, ie, accelerates the gel.
TEMED : N , N , N ', N 'tetramethylethylenediamine, a catalyst that accelerates the formation of a stereostructure of polyacrylamide, ie, accelerates the gel.
After learning so much basic knowledge, we can finally make glue! Oh, no! Slow! Think about it first, how much glue do we have to make? Different polyacrylamide concentrations produce different molecular sieves and will have an effect on subsequent experiments. Common polyacrylamide gel concentrations are: 8% , 10% , 12% (what? Gradient glue, cough, not to mention here), depending on the situation.
Separation gels of different concentrations have different separation effects on proteins of the same range of molecular weights, thereby obtaining corresponding concentrations of separation gels for different molecular weight proteins. After a large amount of experimental data, 8% polyacrylamide gel has a better separation effect on proteins above 35kDa , and has a poor separation effect on proteins below 35kDa ; separation effect of 15% polyacrylamide gel on 15-170kDa protein Ordinary, the protein separation effect below 15kDa is poor; 12% polyacrylamide gel has better separation effect on protein below 45kDa , and the effect on protein separation above 45kDa is poor.
In the Western experiment, most of the proteins have a molecular weight of 20-100 kDa , so the use of a 10% polyacrylamide gel without special requirements can basically ensure the separation of proteins of different molecular weights and the highest utilization of electrophoretic proteins.
Ok, let's make rubber!
The fact that the glue is made is actually a craft work and requires a certain level of proficiency. At the beginning of the rubber production, one or two pieces of waste rubber were produced . You don't have to worry about it. In the same year, BioTNT can make a full day to make a gel that can carry out experiments!
Leakage is often the first problem encountered by every technician friend. Although the glue making tank of the glue is different according to the purchase company, it is generally the same, and the strength of the hand is the most important. For newcomers to make rubber, personally recommend not to rush to make glue, first assemble the gluer, add water to the glass tank, wait for 25 minutes (that is, the time for the glue to solidify), until the water leakage is basically solved, You can start making glue. Generally speaking, there is no water leakage, and the possibility of leaking glue is basically eliminated. In the process of assembling the gluer, it is necessary to pay attention to whether the bottom of the glass plate and the bottom of the glue making tank are flat. Once it is not flat, the possibility of leaking glue is very large. How can we level it? This is the problem of strength, the force can not be too light, the same can not be too fierce, too light can not seal, too fierce is easy to cause deformation, this degree, in the end, you still have to find it yourself! BioTNT 's colleagues also told me that a conditional laboratory can add some agarose gel to the glass tank before making a polyacrylamide gel. Personally, it is also a good suggestion!
Do you think that it will not leak, even if it is successful? No , no , no ! You are still far away! Don't forget, you just got to the rubber step!
Next, I will mention a few points that need attention, and the rest of you will cheer for yourself!
First of all, when preparing liquid glue, you need to be extra careful, do not add wrong or miss something, the mother liquid is also prepared as fresh as possible, do not use it for a long time. The prepared liquid glue needs to be thoroughly mixed. The recommended method is to gently up and down. As mentioned before, AP and TEMED are polyacrylamide coagulants. If you accidentally add it, your glue will not condense. (It is said that there is no such thing as a gel for a week. Anyway, BioTNT is not waiting)!
Secondly, when adding glue, be careful not to add bubbles. SDS is a detergent. It is easy to produce bubbles during the mixing process, and it is very deadly to mix bubbles in the glue! When mixing, the method should be gentle. After mixing, the liquid glue is allowed to stand for about 15 seconds and then added to make the tiny bubbles in the liquid glue float. In addition, when adding glue, you can use two-speed suction and one-speed hit to prevent air from being blown after the glue is finished. At the same time, you should also pay attention to whether the gun head has air bubbles.
Third, the pressure of the glue should be uniform and slow when pressing. Gluing is an indispensable step in the making of glue. The flat separating glue helps to obtain a flat strip. Common glues are isopropyl alcohol, which can also be replaced with ultrapure water. Inadvertently pressing the glue not only can not flatten the separation rubber, but will make the separation rubber uneven, and even dissolve directly into the separation glue, resulting in uneven glue. When dropping the glue, stick the glass plate and slowly add it from left to right to control the volume of each drop.
Fourth, you should pay attention to the time and temperature of the gel. Gel is a chemical reaction that has a certain relationship with temperature and time. In general, when the room temperature is 10-25 ° C, the gel takes 25 minutes. When the room temperature is higher, the time can be reduced as appropriate. On the contrary, when the room temperature is low, it should be appropriately increased. Insufficient gel time will cause the glue to not condense, and too long a time will make it difficult to pull the lane.
Finally, when making the lanes, keep the combs up and down vertically. When inserting the comb, the oblique combing of the comb will make it difficult to pull out and even cause bad glue (may be very large). When pulling the comb, the diagonal pull will open the glass plate, so the sample may leak samples! Secondly, the lane should be kept at a certain height, that is, the liquid separation gel to be added must be sufficient, otherwise the sample overflow can be the nail on the plate when loading! Right, don't forget to add the electrophoresis buffer when pulling the comb!
Ok? Successful? Congratulations! So can the cured glue be stored for a long time? Yes, but not so long, it is strongly recommended to use it on the same day. If it must be stored for a long time, you need to add enough (without colloid) electrophoresis buffer to the refrigerator at 4 °C, pay attention! DO NOT FREEZEN ! If you find that your running buffer is frozen when you use it, you can take it. The remelted glue is very easy to miss when loading. In other words, in the winter, not only should not be refrigerated, but should be kept warm! At the same time, the recommended shelf life should not exceed one week, and the running buffer should be supplemented daily to keep the colloid moist.
After finishing the glue, it finally took the first step of the Long March. Next, it was the official Western experiment. However, the good start is half of the success. With a good glue, the successful experiment will have A good foundation, I wish you all the best in your experiment and get the results you want!
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