Anti - Neu5Gc Antibody Kit Protocol - Western blot
Prepare two identical SDS - PAGE gels .
Load and run samples in loading buffer .
Recommended loading of gel :
Lane 1 : Molecular Weight Marker .
Lanes 2 , 8 , 10 : Blank .
Lanes 3 - 6 : Samples .
Lane 7 : Positive Control .
Lane 9 : Negative Control .
Confirm the presence of proteins on gel by Coomassie Staining Blot proteins to membrane of choice .
Confirm protein transfer by Ponceau or other method of choice .
Block each membrane in 20 ml TBS - T with 200 μl diluted Neu5Gc Assay Blocking Solution , gently rocking at 4°C .
Incubate first blot in Primary Antibody , and one blot in Control Antibody for 2 hours at 25°C or overnight at 4°C with gentle rocking .
Wash blot with 50 ml TBS - T for 5 min at 25°C , gently rocking 5 times .
Incubate blots with optimal dilution of enzyme - conjugated secondary antibody of choice at 25°C for 1 hour .
Wash blot with 50 ml TBS - T for 5 min at 25°C , gently rocking 5 times .
Develop each blot with the appropriate substrate
