Reactive protein bands were visualized using the chemiluminescent

Reactive protein bands were visualized using the chemiluminescent peroxidase substrate (Pierce). 2.9. N-Glycan thoroughly Profile Analysis Samples were first digested into glycopeptides using pepsin, as previously described [19]. From the resulting glycopeptide mixtures, N-glycans were released using peptide N-glycosidase (PNGase) A (Roche, Basel, Switzerland). The released N-glycans were purified using graphitized carbon resin from Carbograph (Alltech, Lexington, MA, USA). Purified glycans were dried and then redissolved in a mixture of 90��L dimethyl sulfoxide (DMSO), 2.7��L water, and 35��L iodomethane for solid phase permethylation using a spin-column method [20]. After the process previously described, the chloroform layer containing permethylated glycans was dried and resuspended in 4��L of a 50% methanol solution.

Then, it was mixed in equal volume with the matrix 2,5-dihydroxybenzoic acid prepared in 1mM sodium acetate solution. The resulting mixtures were applied onto an MALDI MSP 96 polished steel Chip (Bruker Daltonik GmbH, Bremen, Germany) and dried. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was performed in the reflector positive ion mode using a Microflex (Bruker Daltonik). All mass spectra were acquired at a 20kV accelerating voltage using the method recommended by the manufacturer. 2.10. Immunological Analysis of Plant-Derived Recombinant Chimeric Protein Six-week-old female BALB/c mice were purchased from Da Mool Science (Daejeon, Republic Korea) and maintained in a pathogen-free environment.

All mice experiments in this study were approved by the Wonkwang University Animal Ethics Committee in accordance with the guidelines of the Korean Council on Animal Care. Eight-week-old female BALB/c mice (5 per group) were injected 3 times with 1, 5, or 10��g of plant-derived GA733 or mammalian-derived GA733 (R&D systems) in a total volume of 100��L at 2-week intervals. The first and second immunizations were given subcutaneously (s.c.) with complete (Difco, Detroit, MI, USA) and incomplete Freund’s adjuvant (Thermo Fisher Scientific, Roskilde, Denmark), respectively; the third dose was administered intraperitoneally (i.p.) in saline. Blood samples were collected by retroorbital bleeding before the experiment and 10 days after the second immunization; 10 days after the third immunization, mice were bled individually via the retroorbital plexus and sacrificed.

Brefeldin_A 100��L/well of sera (1��g/mL in PBS) were applied to ELISA plates coated with human colorectal cells from lines SW480, SW1116, and SW620, and with human breast cancer cells from line MCF-7 (4 �� 104 cells/well) and incubated for 2h at RT. 100��L/well secondary antibody HRP-conjugated goat anti-mouse IgG (Animal genetics) diluted in PBS (1:8,000) were applied and incubated for 2h at RT.

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>