After estimating the protein concentration of each sample, proteins were digested using a trypsin enzyme to produce proteolytic peptides.
In total, 30 μg of sample proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane.
The sample proteins were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Millipore, USA).
After final centrifugation, the sample proteins were acetone-precipitated into 800 µg or 110 µg aliquots and stored at -80°C.
Equal amounts of sample protein were first treated with Benzonase (Thermofisher) on ice and then resuspended in 1x translation loading buffer (186 mM Tris-Cl pH 6.7, 15% glycerol, 2% SDS, 0.5 mg/ml bromophenol blue, 6% beta-mercaptoethanol).
20 μg sample protein was loaded in the well and was separated by 10% SDS-PAGE.
Approximately, 3 μg of the sample protein was subjected to SDS-PGE, followed by electroblotting on a polyvinylidene difluoride membrane.
Approximately 3 μg of the sample protein was subjected to SDS-PAGE, followed by electroblotting onto a polyvinylidene difluoride membrane.
The glycoprotein sugar moieties of the sample protein were detected in the SDS-PAGE gel using the GelCode Glycoprotein kit (Pierce Biotech., Inc., IL, USA).
Cell lysates were normalized by protein concentration, reduced with DTT and 10 µg of sample protein were loaded into a 4 12% Bis-Tris gel (Invitrogen).
