Good day all, I want to harvest, assay and purify α2,6 and α2,3 sialyltransferases from chicken liver without using the radiolabelling protocol? I will greatly appreciate your prompt response. Thanks immensely.
I have done estimation of α2-6 and α2-3 ST from saliva by ELISA. Article Salivary Glyco-sialylation changes monitors oral carcinogenesis
Linkage specific lectins, sambucus nigra agglutinin (SNA) for α-2,6 and macckia amurensis agglutinin (MAM) for α-2,3 were used for the detection of α-2,6 and α-2,3 linked ST. First biotinylation of SNA and MAM was performed according to procedure of sulpho-NHS Biotinylation kit (Pierce IL). Biotin conjugated
lectins were used for detection of linkage specific STs by ELISA method.
α-2,6 and α-2,3 ST activities were estimated by ELISA method as
described by Hakomori et al. and Yeh and Cummings, respectively with slight modifications.
Hakomori, S.: Glycosphingolipids in cellular interaction, differentiation
and oncogenesis. Annu Rev Biochem 50, 733–764 (1981)
Yeh, J.C., Cummings, R.D.: Absorbance and light based solid phase
assays for CMPNeuAc: Galbeta1-4GlcNAc-Ralpha2,3-sialyl transferase.
Anal Biochem 236, 126–133 (1996)
For isolating from tissues, the liver tissue should be homogenized in PBS (pH:7.4). The whole process should be carried out on ice. The supernatant (cytosolic fraction) be separated by centrifugation at 15,000 rpm in cooling centrifuge. The supernatants should be used for enzyme assays by ELISA.
Enam, it helps if you provide more details about what the problem is. Have you tried using Pubmed or Google Scholar to search exactly for that, and if you did, did you try using the procedures therein?
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