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< > principle"
polyacrylamide gelphoresis (page) is commonly used for separation. < a href" >in protein and smaller moleculesnucleic acid . There are two basic ways: disc-electrophoresis and slab-electrophoresis. Both disc electrophoresis and flat-panel electrophoresis have continuous and non-continuous electrophoresis. Electrophoresis is carried out in a system with consistent electrode buffer, gel buffer, and gel aperture, called continuous PAGE, and electrophoresis is carried out in a system with different electrode buffer, gel buffer pH and gel aperture, called non-continuous PAGE. The non-continuous PAGE separation includes three physical effects: the concentration effect of the sample, the charge effect of electrophoresis separation and the molecular sieve effect. Continuous PAGE, on the other hand, does not have a concentration effect. This experiment introduces non-continuous polyacrylamide gel disc electrophoresis. > ef and equipment
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1. Separation glue buffer (pH8.9) is called Tris 36.3g, lmol/L HCl solution 48ml, plus distilled water 80ml to dissolve it, pH to 8.9, with distilled water to 100ml, placed in a brown bottle, 4c refrigerator storage.
2. Concentrated adhesive buffer (pH6.7) is called Tris 5.98g, lmol/LHCl solution 48ml, add distilled water to 80ml to dissolve it, pH to 6.7, use distilled water to 100ml, in a brown bottle, 4c refrigerator storage.
3. Gel storage liquid (30% monobody crosslinker) called acrylamide (Acr) 29.2g, fork diacrylamide (Bis) 0.8g, distilled water dissolved and fixed capacity to 100ml,filter, the insoluble material filtered out, in brown bottles, Store in a refrigerator at 4 degrees C for 1 month.
4. Electrode buffer (pH8.3) is called glycine 28.8g, Tris 6.0g, plus distilled water to 850ml, pH to 3, with distilled water to 1000ml, 4c refrigerator storage, can be diluted 10 times.
5. The catalyst (10% ammonium persulphate) is said to have taken 0.5g of ammonium sulfate and added 5 ml of distilled water. Pre-use preparation, 4 degrees C refrigerator storage, up to a maximum of 1 week.
6. Accelerator (tethymethyl ethyl glycamine, TEMED) original packaging solution, stored in a refrigerator at 4 degrees C backup.
7. Staining liquid called Kaomas bright blue R-250 (CBBR-250) L 25g, add 50% methanol 454ml dissolved, then add ice acetic acid 46ml, mix well.
8. Fixed liquid 12.5% tychloro acetate (TCA)
9. The semen is taken from 30 ml of ice acetic acid, methanol 125 ml, and the capacity is fixed to 500 ml with distilled water.
10. Save 7% ice acetic acid.
11. 0.05% bromophenol blue
12. The electrophoresis device uses an electronic tube or transistor rectition power supply. Voltage 0-600V, current 0-300mA.
13. Electrophoresis Groove Cylindrical vertical electrophoresis groove.
14. Gel glass tube 0.5 to 0.6cm × 10cm.
15. Small < a href""> beech ,50ul microscope, 10cm long needle or lumbar puncture needle, 5 or 10ml syringe.
1. Prepare the gel column
(1) take the prepared 0.6cm×10cm glass tube, measure from one end to 7cm and 7.5cm, line with a glass crayon, insert the rubber pad and place vertically in the small test tube.
(2) preparation of separation glue: take a small beech, according to table 4-1 to add each liquid, shake well, the total volume of this liquid is 10 ml, gel concentration of 7.5%. Using a 5 ml syringe, a long needle or a capillary dropper to absorb the separation fluid, along the pipe wall into the glass pipe to 7cm dash, carefully cover the gel surface with a layer of distilled water (about 0.5cm high), try to avoid impact on the gel surface. After the gel and water interface can see a line, pour the covered water and suck it dry with a filter strip.
(3) preparation concentrate glue: according to Table 4 to add each liquid, shake well. The total volume is 10 ml and the gel concentration is 3.0%. Inject the gel tube along the wall up to 7.5cm and add distilled water (about 0.5cm high) along the wall. To be polymerized and dumped and dried distilled water, ready to add samples.
2. Sampledserum 0.1 ml, 40% sucrose liquid 0.1 ml and 0.05% bromophenol blue 0.1 ml (as a tracer dye) mixed in a small test tube, with a microscope to absorb 10tq added to the surface of the glass concentrate, equivalent to add serum 3.3ul.
(1) installs the added gel tube in a rubber hole in the electrophoresis slot so that the top of the gel is visible and the rubber pad at the lower end is discarded.
(2) absorb the electrode buffer with a capillary dropper tube and carefully fill the gel glass tube (do not stir the sample layer). Then the electrode buffer is slowly poured up and down the electrode groove, the upper groove should be immersed in the gel glass tube, the bottom groove liquid surface should exceed the lower end of the gel glass tube. Carefully remove possible bubbles in the upper and lower tubes of the gel tube, as this may affect electrical conductivity.
(3) connects the electrode of the upper slot to the negative pole of the electrophoresis instrument, the lower end is positive, and the power is on. The current is adjusted to 1 mAh/tube, and then the current is 2 mAh/tube after the tracer dye enters the separation glue. When the tracer dye reaches about 0.5cm under the glass tube, the power supply is cut off and the electrophoresis is completed.
4. Peel the gel Remove the gel tube and fill the distilled water with a syringe with a 10cm long needle. Carefully insert the needle between the post and the pipe wall, while filling the water while rotating the glass pipe, by water pressure and lubrication to separate the inner wall of the tube from the gel, and then gently pressurized at one end of the tube with the earwash ball, the gel column will slowly slide out of the glass tube.
5. Fix and dye the gel column soaked in 12.5% TCA 0.5 to 1h, and then transferred to CBBR-250 dyeing liquid dyeing l to 2h, or 90 oC water bath dyeing 10min.
6. Decoloring, saving Place the dyed gel column in a 7% ice acetic acid solution and constantly update the ice acetic acid until it is colorless. The whole process takes about 5-7d. Or rinse 3 to 4 times with seven solution, 20 to 30min at a time. Take lcm× l5cm small test tube, containing 7% ice acetic acid solution, soak the gel column in it, add wax seal can be long-term preservation.
(1) slice pumping method: cutting fragments of a specific area, using 10 times the amount of water or 0.1-0.2mol/L NaCl or the appropriate amount of buffer for homogeneity. Overnight pumping at 4 degrees C, centrifugation to take the liquid, choose the appropriate wavelength to determine the absorbent value. This quantitative method is not accurate and can be used as a sample recovery method.
(2) Micro-light density meter: There are currently trace light density meters for direct UV scanning without dyeing, there are light density meters that must be dyed and determined, and ultra-micro-light density meters equipped with microscopic devices. In order to eliminate the speral difference of cylindrical gel, the strip can be put in 7% ice acetic acid for determination.
"Notes"1. Acr and Bis are nerve agents that can be absorbed by the skin, respiratory tract, etc., and should be protected when operating.
2. Acr and Bis are stable at low temperatures and should be stored in brown bottlesatlow temperature (4 degrees C). 30% monobody crosslinker should be in brown bottles, stored in the refrigerator (4 degrees C) can partially prevent hydrolyzing, but can only save l to February. PH (4.9 to 5.2) can be measured to check for failure, failure fluid can not be polymerized.
3. The surface is sealed with distilled water during the process of making the glue in order to prevent the inhibition of oxygen in the air to the polymerization of the gel.
4. TEMED should be sealed to avoid light preservation.
5. Preparation of gel with a glass tube to be soaked with lotion cleaning, such as the glass tube is not clean, after pouring the glue in the pipe wall will appear bubbles.
6. When preparing the separation glue and concentrate glue, the catalyst and accelerator are added to the mixture and polymerized within about 10 to 30min, so the human glass tube should be filled as soon as possible. If the room temperature is too high, in order to prevent excessive polymerization, can be placed in the ice bath operation. If the gel is not polymerized, it is usually due to inaccurate reagent concentrations prepared, or if a reagent is leaking from the gel mixture, it may also be due to the insalinity of the reagent. The mixture should be re-provisioned.
7. This method can be simplified, with the same separation adhesive and the same buffer and pH continuous gel electrophoresis, can also obtain better results.
evaluationpolyacrylamide gel electrophoresis gel strength is good, thermal stability, no electrolysis, high transparency, and gel aperture size adjustable, the required sample size is small, and has a high resolution and other advantages. One of the disc electrophoresis equipment is simple, easy to operate. However, flat electrophoresis can be used for comparative analysis of multiple samples at the same time, and combined with sodium SDS) to form SDS-PAGE, or with isoelectric focus (IEF) with IEF-PAGE two-way electrophoresis, can further improve its resolution and expand the scope of application.
(Responsible Editor: King Kunlun of Great Han) ..
