1. avidin (AV) or streptavidin (SA)?
Although avidin (AV) and streptavidin (SA) have high affinity for Biotin in some applications (avidin,AV) the main problem is its non-specific combination, This is due to the avidin (avidin,AV) sugar chain and higher electrical point structure (pI). Therefore, using streptavidin (SA) it can significantly prevent this non-specificity, or using the same Biotin to retain its property of biotin binding to deglycated avidin (avidin,AV) this non-specificity can also be prevented.
2. Use streptavidin (SA) -biotin as a multifunctional detection system.
Biotin-streptavidin-system uses avidin (AV) or streptavidin (SA) with the help of conjugated antibodies to Biotin, and its detection method can be applied to almost any immunoassay. It can be linked to a variety of cheap, high-quality commercial fluorescent dyes or enzymes. This gives biotinylated antibodies the advantages of signal amplification and increased sensitivity, but also requires optimization of the antibody and conjugate ligation solvent.
3. When using biotinylated antibodies, choose an appropriate buffer system.
It is strongly recommended to avoid FBS in solutions such as blocking buffer to prevent high background and low signal-to-noise ratio (SNR) of fetalbovineserum (FBS) and Biotin. A good substitute is 0.1%-2.0% bovine serum protein fractionV (BSAfractionV). In Westernblot, skim milk powder or casein should be limited to the initial blocking step due to residual Biotin (Biotin can interfere with the detection). “In this case, the antibody solution should be prepared in TBS-Tween compatible with nitrocellulose and PVDF membranes.” If high background is still a problem at this time, the result can be improved with a high purity of 0.2% -6% casein.
4. Reduce the interference of residual biotin in the sample.
Sometimes nonspecific bands may be detected by Westernblot when the Biotin-Streptavidin-System is used. This is a particular fact for tissue preparations and cell lysates, as they may contain enzymes that covalently bind organisms as cofactors. In such cases, it is useful to increase the ionic strength of the buffer solution (~0.5MNaCl) and/or to do Biotin blocking prior to coincubation with the primary antibody. In this case, Biotin removal using streptavidin (SA) agarose microspheres significantly improved the sensitivity of the biotinylation enzyme.
5. Cell surface biotinylation for protein quantification.
Chain mildew Biotin avidin System (Biotin Streptavidin – System) in the noninvasive technology successfully used in chemical tag cell surface proteins, so as to express the cell surface and the effect of swallowing cells (endocytosis), and a series of cytoplasm membrane receptors and antigen directly and accurately quantitative detection. Biotin conjugated to specific receptors and antigens (Biotin) was used to quantify the amount using ELISA or Westernblot. This technique is a rapid and safe alternative to radiolabelation techniques.
Post time: Apr-17-2024