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MB Sample ID: SA257920
| Local Sample ID: | SR14 |
| Subject ID: | SU002665 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | WT/PMM2-CDG |
| Age Or Age Range: | 5-45 |
| Gender: | Male and female |
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Collection:
| Collection ID: | CO002658 |
| Collection Summary: | Briefly, medium was removed from the cells and the cells were washed 3 times with 1 mL Dulbecco PBS containing 0.901 mM CaCl2 (Merck) and 0.492 mM MgCl2 (Merck). Next, cells were incubated with 1 mg/100 mL EZ-Link-Sulfo-NHS-LC-Biotin (Thermo) in Dulbecco for 30 min, RT, shaking. Cells were then washed twice with 1 mL Dulbecco, non-reacted biotin was blocked with 1 mL 20 mM glycine in Dulbecco for 15 min, and cells washed again with 1 mL Dulbecco. Dulbecco was then removed, cells scraped in 200 µL RIPA buffer (with protease inhibitors) and transferred to a fresh Eppendorf tube. Samples were lysed on ice with 3 consecutive freeze-thaw cycles. Further, 30 µL dynabeads streptavidin T1 (Invitrogen) was added to each sample and the samples were washed twice with 500 µL 10 mM ammonium bicarbonate and neuraminidase buffer (100 mM Sodium Acetate Buffer with 2 mM CaCl2 (Merck), pH 5.0). Neuraminidase buffer was removed and 500 µL PBS was added to the beads. 30 µL of prepared mix was added to each sample, and samples were incubated, shaking overnight at 4 °C. Samples were put on a dynabead rack (Invitrogen) and the supernatant was transferred to a new Eppendorf tube and used for protein concentration assay. Beads containing membrane fractions were washed 2 times with 500 µL lysis buffer (2 % IGEPAL (Sigma), 1% Triton X-100 (Sigma), and 10 % glycerol inPBS) and then washed with 1 mL PBS. Samples are centrifuged at 500 rcf, 5 min, 4 °C and PBS was removed. 100 µL neuraminidase buffer containing 0.05 U neuraminidase was added to each sample and samples were incubated overnight at 37 °C, shaking. Next, supernatant was transferred to a new Eppendorf tube and lyophilized at 4 °C. Finally, pellets were resuspended in 100 µL of extraction buffer (80 % MeOH, IS). Sialic acid was measured by LC/MS as described below. El Maven Polly software was used to annotate sialic acid based on m/z ratio and elution time, and determine fractional contribution of glucose in sialic acid. |
| Sample Type: | Fibroblasts |
| Storage Conditions: | -80℃ |
