many bands other than my protein on western... - please help me to explain this... (Feb/18/2006 )
i have expressed my protein in Ecoli and after SDS-PAGE have run western to confirm.....i didnt purify my protein yet (GST) but only have boiled it in 1x SDS loading buffer..protein of my interest is the main very very intense band....but there are other bands also... i have to explain the result in the seminar next week.....and i dont know what to say... thanx a lot
I wouldn't worry about the other bands at the moment. Sounds your western needs a bit of optimisation like a higher diltion of primary and/or secondary antibody to obtain the single signal you are looking for.
Western blot is only as specific as your antibodies are... .
Otherwise the next time you could purify your protein via GST.
Did you have a control, like expression vector without insert and see differences in western blot between vector with insert and without insert?
I guess the probably unspecific bands might occur in the control too... .
thank you ! i did have a negative control without an insert in my western and it doesnt show any bands... i am duliting the sample so so much because if i just ran as i did for SDS-PAGE the protein band appears as black spot and covers everything else....maybe because there is just too much of protein it is digested somehow and many bands appear....
If your negative control is negative on western, and you're getting smaller bands in your experimental lanes, they are either products of proteolytic digestion, or peptides synthesized from truncated RNA, or protein fragments arising as a consequence of boiling, or some combination of all three.
thanxx a lot! i have reviewed some literature about my protein and it seems that it does undergoes digestion after expression.... how can that happen in BL21 strain of E coli? arent they supposed to be protease difficient? ....maybe adding some antiproteases will help?
you can check expression with ptotease difficient BL21 from Amersham, but you can not add any antiproteases because you are intracellular expressing your protein, if you have a lot of protein degradation try to decrese temperature growth and/or IPTG concentration,