......
1. Why does the transfer of protein is needed?
Western Blot is a method in which proteins are transferred to a membrane and then detected using an antibody. For known expression proteins, the corresponding antibody can be used as a primary antibody for detection, and the expression product of the new gene can be detected by the fusion portion of the antibody.
The protein sample separated by PAGE is transferred to a solid phase carrier (for example, a nitrocellulose membrane), and the solid phase carrier adsorbs the protein with a non-covalent bond, and can maintain the type of polypeptide and its biological activity. The protein or polypeptide on the solid phase carrier is used as an antigen, and the corresponding antibody is immunoreactive, and then reacted with the enzyme or the isotope-labeled secondary antibody, and the protein expressed by specific gene is detected by substrate color development or autoradiography.
2. The reason and solution of the incompleted transferance
Reasons
The film is not completely soaked
Use 100% methanol permeable membrane
molecular weight of Target protein is less than 10,000
Select a small pore size membrane to shorten the transfer time
The isoelectric point of the target protein is equal to or close to the pH of the transfer buffer
Methanol concentration is too high
Excessive methanol concentration causes the protein to separate from the SDS, thereby precipitating in the gel, while causing the gel to shrink or harden, thereby inhibiting the transfer of high molecular weight proteins. Reduce methanol concentration or use ethanol or isopropanol instead
Insufficient transfer time
Extended transfer time for thick gels and high molecular weight proteins
3. Preparation method for CAPS (CAS 1135-40-6) buffer
Please see the following table (Both concentrations of buffer and sodium hydroxide are 0.1 mol/L):
pH of CAPS Buffer
Volume （CAPS）
Volume (NaOH)
6.8
250
0
10.0
218.3
31.7
10.5
178.6
71.4
10.8
156.3
93.7
11.2
138.9
111.1
The specific operation of the preparation is: 0.1 mol/L CAPS buffer solution is first prepared in a volumetric flask. During the configuration process, the buffer has to be dehydrated at a high temperature and cooled in a desiccator; 0.1 mol/L sodium hydroxide solution is also formulated at the same time. According to the formula in the above table, a buffer solution corresponding to pH can be prepared, and sodium hydroxide is mainly used to adjust the pH.
Western blotting can be applied to detect target proteins from protein mixtures, quantitatively or qualitatively determine the expression of proteins in cells or tissues, and subsequent analysis of protein-protein, protein-DNA, and protein-RNA interactions. Film transfer is the basis of WB, and its quality is the key to the success of WB. We hope that the above methods can help you succeed in the process of transfor.
Copyright © Suzhou Yacoo Science Co., Ltd. All Rights Reserved
Friendly Links :
online service
86-15062454963
sales@yacoo.com.cn
