Activated sialic acid derivatives for protein derivatization and conjugation
A compound and useful technology, applied in sugar derivatives, peptide/protein components, organic chemistry, etc., that can solve problems such as uneconomical, difficult to achieve reactant concentration, and unfavorable protein stability
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Embodiment 1
[0135] Example 1 - Fractionation of CA (CA, 22.7 kDa, p.d. 1.34) by IEC [ref]
[0136] Load 900ml agarose QFF into an XK50 column and equilibrate with 3 column volumes of wash buffer (20mM triethanolamine; pH 7.4) at a flow rate of 50ml / min. CA (25 g dissolved in 200 ml wash buffer) was loaded onto the column through the injection port at a flow rate of 50 ml / min. Then wash the column with 1.5 column volumes (1350ml) of wash buffer.
[0137] The bound CA was eluted with 1.5 column volumes of different elution buffers (triethanolamine buffer, 20 mM pH 7.4, containing 0 mM to 475 mM NaCl in increments of 25 mM NaCl), and finally all residuals were removed with the same buffer containing 1000 mM NaCl. CA and other residues (if any).
[0138] Samples were concentrated to 20 ml by high-pressure ultrafiltration using a 5 kDa membrane (Vivascience, UK). The buffer of the samples was exchanged with deionized water by repeated ultrafiltration at 4°C. Samples were analyzed for avera...
Embodiment 2
[0139] Example 2: Activation of CA [ref]
[0140] Freshly prepared 0.02 M sodium periodate (NaIO 4 ; 6-fold molar excess over CA) solution was mixed with CA, and the reaction mixture was magnetically stirred for 15 min in the dark. Oxidized CA was precipitated with 70% (final concentration) ethanol, and the mixture was centrifuged at 3000g for 20 minutes. Remove the supernatant and dissolve the pellet with a minimal amount of deionized water. CA was again precipitated by 70% ethanol and centrifuged at 12,000g. The pellet was dissolved with a minimal amount of deionized water, lyophilized and stored at -20°C until use.
Embodiment 3
[0141] Example 3: Determination of the Oxidation State of CA and Derivatives [Reference]
[0142] Quantitative determination of CA oxidation degree with 2,4-Dinitrophenylhydrazine (2,4-DNPH), which interacts with carbonyl compounds to generate a small amount of soluble 2,4-DNPH base hydrazone. Unoxidized CA and oxidized CA (CAO) (5 mg each) were added to 2,4-DNPH reagent (1.0 ml), the solution was shaken and then placed at 37 °C until crystal precipitation was observed [Shrineret.al., 1980] . The (quantitative) degree of oxidation of CA was determined at 630 nm by a method based on the reduction of ferric cyanide ion to ferric ferrocyanide (Persian blue) in alkaline solution [Park and Johnson, 1949]. In this example, glucose was used as the standard.
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