1. Basic information of protein:
CNTN1 (ContactIn 1, also known as F3 protein) is a cell adhesion molecule that belongs to the Ig superfamily. It plays an important role in several physiological and pathological processes such as generation due to its specific cell adhesion function.
The main characteristics of CNTN1 protein are as follows:
1.Molecular weight: approx. 130 kDa.
2.Structural characteristics: Contains multiple Ig-like C2-type domains and abundant glycosylation modifications.
3.Expression pattern: Widely expressed in the central nervous system, peripheral nervous system, and other tissues.
4.Function: Regulates physiological processes such as cell-cell interaction, cell polarity, and embryonic development by binding to other proteins such as Caspr, NRCAM, and Notch1.
5.Catalog No. PA1000-6948
2. Experimental design:
1.Gene cloning:
a.Primers were designed according to the gene sequence of CNTN1, and CNTN1 cDNA was amplified by RT-PCR.
b.Insert CNTN1 cDNA into a fusion expression vector, such as PET-28A(+).
c.Correct clones were verified by digestion identification and sequencing.
2.Recombinant Protein Expression:
a.Transform the fusion vector into an E. coli expressing host, such as BL21(DE3).
b.IPTG was used to induce protein expression, and bacterial expression products were collected.
3.Cell lysis and isolation:
a.Resuspend the bacterial deposits in a lysate containing the appropriate amount of bovine albumin, enzyme inhibitors, and wash buffer.
b.After bacterial lysis, the soluble CNTN1 recombinant protein was isolated by centrifugation.
4.Affinity purification:
a.The CNTN1 recombinant protein is affinity bound to an affinity resin, such as nickel-NTA affinity resin.
b.Elution buffer is used to elute recombinant proteins bound to affinity resins.
c.After elution and concentration, purified CNTN1 recombinant protein was obtained.
5.Protein Identification and Quantitation:
a.SDS-PAGE and Western blot were used to analyze the purity and molecular weight of purified protein samples.
b.The concentration of CNTN1 recombinant protein is determined using the BCA or Bradford method.
6.Test Protein Function:
a.CNTN1 recombinant protein was added to cell culture medium using cell culture and transfection techniques.
b.The effects of CNTN1 recombinant protein on the adhesion and migration ability of cells (such as neurons and cancer cells) were evaluated by cell adhesion and migration assays.
c.Quantitative analysis was performed using cell adhesion and migration metrics such as cell number, cell membrane morphology, and transwell migration assays.