Please help me with the western blot problem - (Oct/03/2007 )
I have been trying the protein expression a lot of times. I always got a lot of nonspecific bands on the western membrane. My protein is His tagged and purified with Ni-NTA beads. At first, I thought it was a purification problem. But after trying with new beads, I think it's probably a expression problem because these nonspecific proteins do bind to nickel beads and are eluted with the target protein. So does anyone know what the problem is? Thanks.
-dv6000t-
QUOTE (dv6000t @ Oct 3 2007, 11:42 PM)
I have been trying the protein expression a lot of times. I always got a lot of nonspecific bands on the western membrane. My protein is His tagged and purified with Ni-NTA beads. At first, I thought it was a purification problem. But after trying with new beads, I think it's probably a expression problem because these nonspecific proteins do bind to nickel beads and are eluted with the target protein. So does anyone know what the problem is? Thanks.
How much beads did you use for what volume of culture/expected amount of recombinant protein? Ni-NTA.Beads tend to have lots of unspecific binding if used in excess. With purifying HIS-tagged proteins it is better to use little bead-volume as possible.
The reason for unspecific binding might be that once the HIS-tag has bound to the beads and the beads have still free binding capacity, also other HIS moieties of other proteins (triple to quadruple HIS in a protein) can bind to the beads with lower affinity. Therefore as mentioned above, use little bead volume as possible. Then only the HIStagged protein will bind.
To increase the yield of the purification, it´s better to purify the flowthrough again via Ni-NTA.
-Dukes-
QUOTE (Dukes @ Oct 3 2007, 11:22 PM)
QUOTE (dv6000t @ Oct 3 2007, 11:42 PM)
I have been trying the protein expression a lot of times. I always got a lot of nonspecific bands on the western membrane. My protein is His tagged and purified with Ni-NTA beads. At first, I thought it was a purification problem. But after trying with new beads, I think it's probably a expression problem because these nonspecific proteins do bind to nickel beads and are eluted with the target protein. So does anyone know what the problem is? Thanks.
How much beads did you use for what volume of culture/expected amount of recombinant protein? Ni-NTA.Beads tend to have lots of unspecific binding if used in excess. With purifying HIS-tagged proteins it is better to use little bead-volume as possible.
The reason for unspecific binding might be that once the HIS-tag has bound to the beads and the beads have still free binding capacity, also other HIS moieties of other proteins (triple to quadruple HIS in a protein) can bind to the beads with lower affinity. Therefore as mentioned above, use little bead volume as possible. Then only the HIStagged protein will bind.
To increase the yield of the purification, it´s better to purify the flowthrough again via Ni-NTA.
Thanks! This is an important clue. But the weird thing is that even I used the bacteria for western blot analysis before the purification step, I still got the same result. I am so frustrated now. Does anyone know what the hell these nonspecific bands are?
-dv6000t-