Hello, I am working on the extraction from Total Protein Soluble and then purification of protein expressed in Nicotiana benthamiana leaves agroinfiltrated with Agrobacterium. Could you suggest me some related protocols? Now I have had a low yield.

I am trying the next protocol using Ni+2 affinity chromatography:

Extraction Buffer:

Sacarose 250mM -------- 1.25mL Sacarose 1M

HEPES 50mM -------------500uL HEPES 0.5M pH 7.2

EDTA 1mM ----------------12uL EDTA 0.428M

20mM B-mercaptoetanol ---------- 200uL B-mercaptoetanol 0.5M

0.06% P/V de L – cisteine --------- 0.00316g

MgCl2 1mM ----------------50uL MgCl2 1mM

0.6% P/V de PVP ---------------- 0.03g PVP

Triton 2% ---------------------- 100uL Tritón

PMSF 1mM ------------------ 100uL PMSF 50mM

Washing Buffer: (PBS pH 8,0, 10 mM Imidazole, PMSF al 1 %) “Store 2-4ºC”

Elution Buffer: (PBS pH 8,0, 250 mM imidazole, PMSF al 1 %) “Store 2-4ºC”

Could I use a sonicator preview to centrifugate the samples with the extraction buffer?

Thank you