hello all,
I am trying to produce His-Human-TGFB1 in tobacco leaf tissues using agrobacterium mediated transient expression system. The Purification is based on His-Tag Ni-NTA system. Protein is being purified but 60-80% of eluted protein consists of plant protein.
Conditions are as follows:
Note:His-TGFB1 is hydrophobic in nature; a Dimer; commercially available protein is recommended to reconstitute in Citrate buffer pH=3.0 or 4mM HCL
1. extraction buffer: 50mM Tris or 50mM PBS (pH=6.0), 300mM NaCl, 0.2% tritonX100, 1XPI, 5% glycerol, 10mM Imidazole, No DTT.
2. washing buffer is same as above except No PI, DTT,Triton. Imidazole con 25mM.
3. Elution is in same buffer with 300mM imidazole and no PI, DTT, Triton etc
I am looking forward for the suggestions. All are welcomed.
Thanks in advance
thanks Yi Wan
and Zhe Zeng. I will take these suggestions into consideration.
@Aditya Soni: In general, magnetic agarose beads should be preferred, when non-specific binding is an issue. In this field there are some suppliers, including Cube Biotech, which sells high capacity Ni-NTA MagBeads for a reasonable price. And, in addition to that, INDIGO Ni-MagBeads, which are tolerant against EDTA and DTT. There is a Korean distributor, too...
https://cube-biotech.com/products/magnetic-beads/his-affinity-magbeads/indigo-ni-magbeads/
https://cube-biotech.com/international-distributors-and-partners
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