For protein deglycosylation, 20 µl of tick saliva was incubated under denaturing conditions with a cocktail of α-Gal-free glycosidases (PNGase F, 36 kDa; α-(2-3,6,8,9)-neuraminidase, 69 kDa; O-glycosidase, 180 kDa; β(1-4)-galactosidase, 350 kDa; β-N-acetylglucosaminidase, 140 kDa) that removes both asparagine-linked (N-linked) and serine/threonine-linked (O-linked) oligosaccharides using the EDEGLY enzymatic protein deglycosylation kit (Merck & Co., Inc.) and following the manufacturer’s recommendations [34 (link)]. After deglycosylation, the tick saliva sample was diluted 1:20 in PBS for filtration first through an Amicon 50 kDa unit (Merck & Co., Inc.) to remove deglycosylases except PNGase F, and then the flow-through with tick salivary proteins (most proteins had less than 50 kDa; Fig. 2 A) was filtered through the Amicon 3 kDa (Merck & Co., Inc.) to remove buffer and retain proteins.
Amicon 50 kda unit
Manufactured by Merck Group
The Amicon 50 kDa unit is a laboratory filtration device designed for the concentration and purification of macromolecules, such as proteins, enzymes, and antibodies, with a molecular weight of 50 kDa or greater. The unit employs a semipermeable membrane to selectively retain the desired molecules while allowing smaller components to pass through.
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Lab products found in correlation
2 protocols using amicon 50 kda unit
1
Protein Deglycosylation of Tick Saliva
2
Protein Deglycosylation of Tick Saliva
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For protein deglycosylation, 20 µl of tick saliva was incubated under denaturing conditions with a cocktail of α-Gal-free glycosidases (PNGase F, 36 kDa; α-(2-3,6,8,9)-neuraminidase, 69 kDa; O-glycosidase, 180 kDa; β(1-4)-galactosidase, 350 kDa; β-N-acetylglucosaminidase, 140 kDa) that removes both asparagine-linked (N-linked) and serine/threonine-linked (O-linked) oligosaccharides using the EDEGLY enzymatic protein deglycosylation kit (Merck & Co., Inc.) and following the manufacturer’s recommendations [34 (link)]. After deglycosylation, the tick saliva sample was diluted 1:20 in PBS for filtration first through an Amicon 50 kDa unit (Merck & Co., Inc.) to remove deglycosylases except PNGase F, and then the flow-through with tick salivary proteins (most proteins had less than 50 kDa; Fig. 2 A) was filtered through the Amicon 3 kDa (Merck & Co., Inc.) to remove buffer and retain proteins.
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