I normally do IP then western blot but my problem is i got an extra band (my band in 52KDa and the extra band 30 KDa)
i followed thr folowing protocol
1- add 5ul of mouse anti human antibody (IP) incubation over night
2- add 20 ul proten G agarose
3-collect immunoprecipitates then wash pellet 4 times with PBS
4- dilute pellet with 30 ul lysis buffer and add sample buffer , boil , centerfuge to pellet the agarose beads
5- SDS page analysis of the supernatant
6-Transfer to nitrocellulose membrane.
8- incubate membrane with mouse anti human antibody( primary)
9- wash the incubate with rabbit ant mouse antobody HPR (secondary)
12- X ray film
please help me by answer me why i got an extra band?????
hi I am PhD student (Immunology) i am facing problem in lab when i isolat Monocyte from human blood using CD14 beads and generate them to dendritic cells most the cells die during incubation or lose them during the washing step, i need helo in solving that problem because i dont knoe the cause of that . Please