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WB experiment: What'...

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WB experiment: What's wrong with a blank on the membrane?


Hengyuan Biotechnology mainly engages in scientific research reagent products and currently has over 500000 types of gene products for various species; More than 200000 types of recombinant proteins; There are over 30000 types of polyclonal antibodies from dozens of species and hundreds of high-quality monoclonal antibodies. Hengyuan brand has independently developed ELISA kits, which now cover over 20 species and thousands of products, involving more than ten research fields such as tumors, hormones, autoimmune, cardiovascular, and metabolism. This week, the editor brings you the results of the WB experiment - a blank on the membrane. What is the reason for this? Let's take a look together!


1、 The glue and film are reversed: If the position of the glue and film is reversed during the transfer process, the protein will transfer from the glue to the buffer instead of reaching the film. For standard transfer printing, the gel should be close to the negative pole of the sandwich, and the film should correspond to the positive pole.


2、 Low efficiency of membrane transfer: the efficiency of membrane transfer is affected by many factors, including the size of protein, the percentage of acrylamide in gel, electric field strength, transfer time and pH value of buffer solution. Generally speaking, the larger the protein, the slower the transfer. The best way to transfer large proteins is to use a high electric field intensity. However, small proteins exposed to high electric field intensity for a long time may emit a transfer film. The way to avoid this problem is to use 0.2 μ Transfer printing on PVDF film with an aperture of m. If the isoelectric point of the protein is close to the pH value of the buffer, then the protein carries very little charge and hardly moves in the electric field. If your target protein is strongly alkaline, you can use carbonate (pH 9.9), CAPS (pH 11), and acidic buffer.


3、 Reagents left for too long or stored under incorrect conditions: Antibodies will slowly degrade, and if repeatedly frozen and thawed, they will degrade quickly. The substrate should be stored at -20 ℃.


4、 Antibody impurity or low titer: The concentration of primary antibodies varies greatly, and the dilution of primary antibodies should be determined based on experience. The general principle is to dilute the serum or tissue culture supernatant with a ratio of 1:100 to 1:1000, and to dilute the serum of ascites or hyperimmune animals with a ratio of 1:1000 to 1:10000. The dilution of the secondary antibody at the imprint level of Bio Rad is 1:3000.


5、 Enzyme inactivation: Sodium azide is an inhibitor of horseradish peroxidase. Do not use any reagents containing sodium azide in HRP color Western blot. Sodium azide can be used in alkaline phosphatase bound antibodies without any side effects. Additionally, only distilled deionized water can be used.


6、 Washing with Tween-20: Tween-20 may interfere with certain antibody antibody interactions or wash away target proteins on PVDF membranes. Therefore, except for the first washing after sealing, Tween-20 is not used during other washing processes.


7、 The detection system lacks sufficient sensitivity: ensuring that the amount of protein loaded is within the sensitivity range of the detection system.


For more experimental information, please consult our sales representative. Shanghai Hengyuan Biotechnology Co., Ltd. has its own laboratory and a professional technology research and development team, with long-term
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