To evaluate the N-glycans on AGP, we performed a lectin-array analysis. Briefly, 20 μL of serum was diluted by 80 μL of probing buffer. Diluted serum was added to the well of recombinant lectin array chip (Rexxam Co. Ltd., Osaka, Japan). After 70 mins incubation and washing twice, 100 μL of probing buffer containing 1 μg/mL biotinylated anti-AGP antibody (EPR5605) (Abcam, ab134042) was added to well. After 60 mins incubation and washing twice, 100 μL of probing buffer containing 1 μg/mL Cy3 labeled streptavidin (GE-Healthcare, Buckinghamshire, UK) was added to the well. After 30 mins incubation and washing twice, the chip was scanned by utilizing Bio-REX Scan 200 evanescent fluorescence scanner (Rexxam Co., Ltd.). Mean fluorescence intensity of lectin reactive glycan carrying Igs was normalized by total Igs level.
Cy3 labeled streptavidin
Manufactured by GE Healthcare
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Cy3-labeled streptavidin is a fluorescent protein complex used in various laboratory applications. Streptavidin has a high affinity for the biotin molecule, and the Cy3 fluorescent dye is covalently attached to the streptavidin for detection purposes.
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5 protocols using cy3 labeled streptavidin
1
Lectin-Array Analysis of AGP N-Glycans
2
Cytokine Quantification Assay Protocol
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HMDS was purchased from Shin-Etsu Chemical Co. Ltd. (Tokyo, Japan). Goat-derived Cy3-labeled anti-mouse IgG antibody and Cy3-labeled streptavidin were from GE Healthcare Bio-Sciences Corp. (Piscataway, NJ, USA). Mouse IgG and rabbit IgG were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Interleukin 2 (IL-2) Human Antibody Pair including anti-IL-2 antibody and biotin-labeled anti-human IL-2 antibody was purchased from Invitrogen Corp. (Camarillo, CA, USA). Other reagents were of analytical grade from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Black 96-well polystyrene microplates with flat bottoms consisting of six 16-well modules (F16 MaxiSorp, FluoroNunc) were purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA).
3
Differential Protein Expression in CIC vs. Non-CIC
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To investigate the differential protein expression of CIC vs non-CIC, analysis was performed using a Cell Signaling Phospho Antibody Array (PCS300, Fullmoon Biosystems, Sunnyvale, CA, USA) in e-biogen (Seoul, Republic of Korea), following one of the typical methods of analysis in e-biogen. Whole cell lysates were extracted by RIPA buffer containing protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). After protein extraction, glass slides for microarrays were incubated on a shaking incubator for 45 min after coating with 304 of total or phospho antibodies. After treatment with a blocking solution, the slides were thoroughly washed with distilled water, after which bound-biotinylated proteins were detected using Cy3-labeled streptavidin (1:1000 dilution, GE Healthcare, Little Chalfont, UK). After 20 min of incubation, the slides were washed, completely dried, and then scanned with a GenePix 4100A scanner (Molecular Devices, Sunnyvale, CA, USA). The scanned images were quantified using GenePix 7.0 software (Molecular Devices).
4
Lectin Microarray for IgA1 Glycosylation
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Antibody-overlay lectin microarray was performed as described previously [12] (link), [13] (link). Purified IgA1 was diluted to 80 µL with PBS containing 0.1% Triton X-100 (PBS-Tx) and applied to the lectin microarray. After incubation at 20°C for 18 h, 1 µg human serum polyclonal IgG was added to the array followed by 1 h of incubation. The reaction solution was discarded, and the array was washed with PBS-Tx. A biotinylated anti-IgA1 mAb (400 ng) diluted in 80 µL PBS-Tx was applied to the array followed by incubation at 20°C for 1 h. The array was washed with PBS-Tx and then incubated at 20°C for 30 min with 400 ng Cy3-labeled streptavidin (GE Healthcare) diluted in 80 µL PBS-Tx. The array was rinsed with PBS-Tx and scanned by an evanescent-field fluorescence scanner (GlycoStation; Moritex, Tokyo, Japan). All data were analyzed with Array Pro analyzer (ver. 4.5). The net intensity was calculated by subtracting the mean background value from the mean signal intensity of three spots per lectin. The obtained signal intensities are available as Table S1 , S2 ,S3 as well as at figshare.com (10.6084/m9.figshare.894402).
5
Lectin Microarray Analysis of Sialylated MUC1
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The antibody-overlay lectin microarray was performed as described previously. 22, 26 In brief, immunoprecipitated sialylated MUC1 from the protein extraction samples described above was diluted with PBSTx and then applied to the lectin array slide, LecChip (GlycoTechnica, Yokohama, Japan) containing triplicate spots of 45 lectins (Supplementary Figure S2). After overnight incubation at 20 °C, human serum polyclonal IgG (20 μg) was added and incubated for 30 min at 20 °C. After the slide was washed three times with PBSTx, 60 μl of the biotinylated MY.1E12 solution (100 ng) in PBSTx was applied to the array and then incubated for 1 h at 20 °C. After the slide was washed three times with PBSTx, 100 ng of a Cy3-labeled streptavidin (GE Healthcare UK, Little Chalfont, UK) solution in PBSTx was added to the slide and then incubated for 25 min at 20 °C. The slide was washed with PBSTx and scanned with an evanescent-field fluorescence scanner (GlycoStation Reader 1200, GlycoTechnica). All data were analyzed with Array-Pro Analyzer, version 4.5 (Media Cybernetics, Bethesda, MD, USA). The net intensity of each spot was calculated by subtracting the background value from the total signal intensity of three spots. The mean lectin signals of triplicate spots were normalized to the mean value of 45 lectins immobilized on the array as described previously. 33
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