Q1: The glue between the two fast glass plates is why the glue is always uneven?
1) The glass is not washed.
2) The addition amount of ammonium persulfate and TEMED is not suitable, and the amount added is relatively large. If the gel is solidified too fast, the gel will be uneven, and the molecular clone will be doubled at most.
3) After the addition of the reagent, it is not well shaken, resulting in too high concentration of the polymerization agent in some parts, and the polymerization is relatively fast, causing the rubber to be uneven.
4) Pay attention to the technique and add it evenly.
5) After filling, the rubber surface should be pressed with water or n-butanol.
6) The edge glue is not easy to be polymerized completely. Immediately after filling the glue, it should be covered with anhydrous n-butanol. The lower the concentration, the more glue should not be used, and the other can also be used in the topping reagent such as n-butanol. Add a little AP to increase the cohesive strength of the edge glue.
7) Temperature is also an important factor affecting the polymerization of the glue. It may be placed in a 50-degree oven for the gel to solidify more quickly. Uneven heating will also cause uneven polymerization.
Q2: Why is the glue always leaking?
1) Wash the glass, strips and other accessories after each electrophoresis.
2) It is important to avoid damage to the edge of the glass (in contact with the strip), especially where it comes into contact with the strip.
3) The key is to find a flat desktop, so that the bottom of the two glass is very close.
4) The strip must be dry. After running the electrophoresis, the strip is placed on the test bench to dry.
5) The following strips should be careful not to age. If there is a crack, use a plastic wrap to put it on top. It can also effectively prevent leakage of glue or vice versa.
6) The glass is easy to cause small gaps during use, which may result in the seal not being completely sealed. After the shelf is installed, a thin layer of Vaseline is applied to the bottom edge of the glass, and there are many points on both sides (easy to leak from both sides) ) This will not leak, even if it has been used for a long time, there are no problems with many small gaps of glass, and then transferred to the electrophoresis tank, remove the seal, and dry the Vaseline on the bottom. (Note, be sure to wipe clean, especially a small amount into the cavity between the two glass, because Vaseline is not conductive, it will affect the effect of electrophoresis.)
7) You can put some paper under the sponge pad to make it more closely attached to the glass. Or seal it with agarose at the bottom of the glass.
8) Because the two glass plates are not perfectly aligned, the bottom is not in the same plane, so the seal is not tight, and it will not leak after realignment. Afterwards, just align the two glass plates on the horizontal table and clamp them again. After feeling the hand on the same plane, put it on the glue holder and press it on the seal and tighten it. Pay attention to the uniform force on both sides, generally no more leakage.
9) Put the two slides on the clip first, do not tighten them, put them on the shelf, align the bottoms of the two slides, clamp the two slides, then remove them and put a gasket under them. It will not leak glue.
Q3: Does the band run narrower than normal?
1). It may be because the gelation is not uniform, the polymerization is not very good, try to mix when filling the glue.
2) It may be related to pulling the comb. Pulling the comb should be quick. When washing the sample hole, be careful to avoid twisting the sample tape. The glue is equipped with a gun to blow a few times and then glue, and the indicator is running. When the two layers of glue meet in a line, increase the voltage.
3) It may be a problem with the sample. If the salt concentration is high, the other strips will be squeezed, resulting in a narrow strip width. For the same reason, the sample will be slow in the gel, causing the strip to go. Slower.
4) The common reason is that the amount of sample loading per hole is not uniform, and the amount of sample in each hole should be consistent.
5) It may be an electrode buffer, or it may be a gel buffer, and the buffer is updated.
Q4: Why do you have the effect of “smile†and “smile�
1) "Smile" is due to uneven cooling during filling and poor cooling in the middle part, resulting in different mobility of molecules in the gel. This is often the case with thicker gels and vertical electrophoresis. "Flip smile", also known as "frowning" phenomenon, is often caused by improper installation of the electrophoresis tank during vertical electrophoresis, especially when there is a bubble at the bottom of the sandwich composed of gel and glass plate or the gel polymerization near the septum is incomplete. Will produce this phenomenon
2) The book says that “smile†is due to the uneven condensation of the whole glue. The middle part and the two ends are heated differently, which may cause “down smileâ€. It may be because the gel at the two ends is not good. After adding APS and TEMED It should be mixed evenly.
3) The salt concentration in the sample is too high? The sample amount is too much or the sample amount per hole is not uniform? The electrophoresis voltage is unstable or too high.
Q5: What should I do with a lot of bubbles when making Western-blot?
1:) First of all, the glass plate must be cleaned, washed and washed, washed, then washed with double distilled water, and dried. When adding the glue, slowly add the ingredients along the wall of the tube. When mixing, gently shake it by hand. If you blow with a gun, be slow and don't hit the head.
2:) When mixing with glue, it should be light, try not to produce bubbles, fast when glued, and the gun does not hit the end. After the separation gel is added, it is sealed with a double distilled water and allowed to solidify. Even if there are air bubbles on the liquid surface during sizing, it will float up after adding water. After separation and gelation, pour off the water inside, blot dry with absorbent paper, add concentrated glue, and insert the comb quickly.
3:) When pouring the glue, slowly add it along the side with a 1ml gun, do not hit the head, so as not to bring in bubbles!
4:) After the glue is placed in a small beaker, tilt the electrophoresis tank slightly, and place the mouth of the beaker against the edge of the glass, and pour it directly. The speed is fast and it is not easy to generate bubbles. Try it.
5:) When using a double steamed water seal, it is recommended to add water with a 200ul gun, so that the pressure is small, so as not to wash the glue!
6:) Domestically, you can only add slowly, don't add bubbles. If you add it, carefully knock the entire board on the table to make the bubbles float up.
7:) The bubbles in the separation gel are related to the insertion of the comb, and the vertical insertion is easy to generate bubbles, and it is not allowed to be excluded. The comb is inserted at an angle of 5-10 degrees, and even if bubbles are generated, the bubbles can be escaping from one side by gently shaking the comb.
8:) There is also a reason for the bubble making of the glue, that is, the liquid of the glue is all stored in the fourth degree. The temperature difference and the exothermic heat of the gel polymerization reaction at room temperature, the tiny bubbles in the liquid are solidified. The glue will also be magnified into larger visible bubbles, which is more likely to occur at higher room temperatures in summer. The method to avoid is to dispose the part of the rubber composition except the catalyst and let it stand at room temperature for a while, and then reduce the temperature difference and then add the catalyst to make the glue.
Q6: The background is too high
1:) Closed.
2:) Cross reaction of primary antibody or blocking solution with membrane protein.
3:) Some impure substances in primary or secondary antibodies can also cause background.
4:) This may be the case if the antibody concentration is too high, or if the incubation time is too frequent.
5:) Membrane or buffer contamination
6:) The film is not completely infiltrated
7:) Closed time control
8:) The film is not washed clean
9:) Color development
Q7: More miscellaneous bands
1) There are many heteroproteins, and the target protein changes after treatment.
2) Antibody specificity is not strong
3) Reduce the incubation time and concentration of the first and second antibodies
4) Increase the number of washings
5) Reasonable sealing method
6) Confirm whether the secondary antibody cross-reacts with the sample antigen
7) Reduce substrate color development time and exposure time
Q8: No purpose strip
1) Short electrophoresis time, not separated from similar molecular weight proteins
2) Whether the protein is expressed or not
3) Maybe the film is turned out
4) Less loading
5) Antibody concentration
6) The target protein is cleaved
7) Problems with luminescent liquid and developer
8) Shorten the closure time and washing time
9) Selection of the concentration of glue
Q9: Large molecular weight proteins are not well transferred to the membrane
1) The pore size of the membrane is too small
2) The film voltage and current are too low
3) The film transfer time is too short
4) It may be too much glue concentration
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