Preparation of CAPS in high purity with low cost

Good's buffers are zwitterionic buffers, which are generally sulfamic acid-based organic compounds, their pH values are relatively stable and does not react with various enzymes and chemical reagents. It is mainly used in the field of biology and medicine. CAPS (N-Cyclohexyl-3-Aminopropanesulfonic Acid) is one of the commonly used Good's buffers in separation of basic drugs by enzymatic chemistry and HPLC. Therefore, its preparation is very important. In order to reduce the reaction conditions, it is generally carried out under the conditions of a catalyst and infrared light irradiation in the production process, while the production cost is increased. And at the same time, the purity of the preparation is difficult to be improved due to the instability of the reaction conditions. This article describes a low-cost method for preparing high purity CAPS.


The preparation process includes:
(1) Preparation of catalyst cerium light rare earth doped nano titanium dioxide: titanium powder and cerium light rare earth are mixed at a weight ratio of 10:1, calcined in a muffle furnace at a temperature of 650°C for 15 h, and then dissolved in distilled water. It was shaken in a water bath, mixed by shaking at 65°C for 60 min, and finally filtered and dried.
(2) The sulfite, bisulfite and propenol are reacted under the special catalyst to prepare 1,3-propanesulfonate, the reaction temperature is 65~90°C, the pH is 6~6.5, and the reaction time is 4~12 hours, the reaction molar concentration ratio is: propenol:bisulfite:sulfite = 1:1.1~1.7:0.1~0.6;
(3) reacting the obtained 1,3-propanesulfonate with carbonic acid at a temperature of 30~50°C for 1~2 h to obtain 1,3-propanesulfonic acid;
(4) The obtained 1,3-propanesulfonic acid and cyclohexylamine are reacted in a water bath at a temperature of 70~90°C for 2~5 h to obtain a crude product of 3-cyclohexylamine-1-propanesulfonic acid;
(5) The crude product of 3-cyclohexylamine-1-propanesulfonic acid is dehydrated by molecular distillation technique at a temperature of 140~150°C and a pressure of 13~266 Pa;

(6) Washing with acetone for 3~7 times to remove cyclohexylamine (the ratio of acetone-washed acetone and crude 3-cyclohexylamine-1-propanesulfonic acid is 5:8), and finally washing 3-cyclohexylamine with distilled water for 3~7 times, and 500 mL of distilled water was used each time to obtain purified 3-cyclohexylamine-1-propanesulfonic acid.


The nanometer titanium dioxide catalyst with rare earth cerium is high in catalytic performance, and the reaction can be carried out without irradiation of infrared light; the sulfite and bisulfite are widely used, and are easily removed in the reaction; during the washing process, the PEEK can be effectively cleaned to ensure maximum acetone savings and lower production costs.


Reference

Yuan Xuesheng. Preparation method of 3-cyclohexylamine-1-propanesulfonic acid. 2016.11.16, CN106117088A


Edited by Suzhou Yacoo Science Co., Ltd.

The incompleted transfer of protein in WB? CAPS may help you!

We always encountered various problems in Western Blot (WB):

•  Incomplete transfer of protein?
•  High background?
•  No positive bands?

......

Below we will give corresponding solutions to the possible reasons for the problem of insufficient transfer of protein, and hope it can help you succeed in the experiment.

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	Solutions
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	Try using other buffers such as N-Cyclohexyl-3-Aminopropanesulfonic Acid (CAPS) buffer (pH 10.5) or using low pH buffers such as acetate 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.

Edited by Suzhou Yacoo Science Co., Ltd.