I've cloned a gene which size was around 800bp in pet 32 vector next to n-terminal histagg to further purify the protein. when I expressed the protein there was a big band around 50 kda + my protein of interest which was around 29kda and others bands on the gel. After purification it remained my protein of interest + that band around 50 kda, what maybe the problem?As I know histagg can only add a small size modification.
Hi Abire.
There can be many reasons for this extra band.
As a masspectrometrist I would start by cutting out these bands and analyse with mass spectrometer to find out what this protein is.
It could be a dimer of your protein, or protein bound to you protein during purification or a protein with affinity to Ni even though it is not His-tagged.
I would:
1. check what protein this is with mass spectrometer.
2. If not a dimer of your protein, you can try to add urea to you His-tag purficationprotocol. That will denaturate you protein and all connections to other proteins (and then you need to slowly get rid of the urea for the protein to refold.
3. If it is an unspecific binding of another protein with affinity for Ni one could try to increase the concentration of imidazole in the wash or sample solution to compete with the unspecific binding.
Sometimes it is nececsary to have another purification step in the protocol, like ion-exchange, after the His-tag purification.
A negative control without overexpression can be added. After running the gel, Coomassie blue staining is used. After the comparison, it is determined whether the 50kda protein is a cell-inherent protein or if you are over-expressed. If it is an intrinsic protein, it may be caused by non-specific adsorption of proteins. If it is overexpression, it may be a dimer or a downstream protein.
i agree with Gudrun Rutsdottir i also suggest you to change the concentration of imadazole in your washing and elution buffer during purification with affinity chromatography.
Thankyou to you all, it is just the mcherry protein that I'm trying to purify and I couldn't use urea because I want to use microscope, but I m wondering if it is possible that this overexpression could be due to others tag in the vector? because the pet32 vector has many others tag beside histagg.
What resin / purification product do you use for the his tag purification?
Or more spicifically which metal Ion?
This page has a figure on it, that shows the specificity of a metal Ion during protein purifications and its corresponding protein yield.
https://cube-biotech.com/products/purification-resins/special-imac-resins/#
Maybe switching to an Ion like Cobalt like Co-NTA resin is enough to get rid of that extra band.
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