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The phosphomannan core nanoparticle prepared and characterized in my earlier studies were
lyophilized in order to couple them to the DVS activated seralose matrix and were coupled to
the same, with a long term objective to use this matrix for the purification of mannose 6-
phosphate receptor proteins.
Isolation of Cathepsin D from Unio homogenate and Beta D Glucuronidase from goat
liver tissue.
Unio tissue was homogenized and passed through Lactose affinity chromatography column and
the lactose specific lectin was isolated, the unbound fraction of the homogenate was processed
further and was passed through Pepstatin A affigel column. The bound cathepsin D protein was
eluted from the column using elution buffer. The eluted fractions were concentrated by amicon
concentrator. The sample will be used for nanoparticle preparation and characterized.
Goat liver tissue was homogenized and was processed further to isolate another lysosomal
enzyme Beta D Glucuronidase
Initially the homogenate was passed through Phenyl sepharose column and eluted fractions
were assayed for enzyme activity. Active fractions were pooled and passed through DE-52
(anion exchanger). Unbound sample was removed by extensive washing with column buffer.
Gradient elution was performed for enzyme purification, initially 50mM NaCl in column buffer
was used for elution and then 100 mM and finally 200 Mm
I could find the enzyme activity in 50mM fractions, active fractions were pooled and
concentrated.
To further purify the protein G-200 sephadex matrix was packed in 100ml column and the
partially purified sample was passed through the matrix. The eluate after the void volume were
carefully collected and assayed for enzyme activity and analyzed by SDS-PAGE as shown
below, the band pattern of the enzyme has to be further confirmed
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Preparation and characterization of galactan and galactomannan polysaccharide (from
Strachnous seeds) nanoparticles with Mohammad
Programme of work for next semester
Preparation of nanoparticles for these lysosomal enzymes one prepared from the
invertebrates and one from the vertebrates, and carrying out similar studies as
described in my earlier reports, would further strengthen the role of the receptor in
nanoparticle delivery in specific cell types.
Preparation of antisera for PMC and PMP-BSA in rabbits to quantify the
nanoparticles developed in the study.
In vitro blotting techniques for recognition of nanoparticles by receptor/antibody to
receptor blots Isolation plant polysaccharides (from Moringa oleifera seeds) and preparation of
nanoparticles and characterization of the same.