Thermo Scientific Y-PER 6xHis Fusion Protein Column Purification Kit uses Y-PER Yeast Protein Extraction Reagent and nickel-IDA (Ni-IDA) agarose columns to extract yeast cells and purify His-tagged fusion proteins.
The kit provides purification of His-tagged fusion proteins from S. cerevisiae and other yeast cells. The protocol that accompanies the product has been optimized to give a high yield of pure histidine-tagged fusion protein in the least amount of time. From 1 to 5g wet yeast cell paste containing overexpressed soluble His-tagged protein, the default procedure produces 3 to 25mL of lysate and 1 to 12mg of purified His-tagged protein. The kit uses Y-PER Yeast Protein Extract Reagent to gently and efficiently lyse yeast cells to extract soluble proteins. The Y-PER Lysate is loaded directly on the pre-packed nickel agarose column, which binds to the His tag. After washing to remove non-bound components of the lysate, the purified His-tagged protein is eluted with an imidazole buffer. Yield and purity greatly depend on the soluble expression level and nature of the recombinant protein.
Highlights:
Yeast (or bacteria) – kit is effective for Saccharomyces cerevisiae and Schizosaccharomyces pombe, as well as Bacillus subtilis and E. coli
Capacity – purify >10mg of overexpressed His-tagged fusion protein from up to 6g wet yeast cell paste
Purity – yields histidine-tagged fusion protein free of cellular contamination (Figure 1)
Speed – progress from lysis to purified protein in less than three hours
Easy and efficient cell lysis – no sonication required; obtain complete cell lysis and extraction of soluble protein with Y-PER Yeast Protein Extraction Reagent
Ready-to-use columns and reagents – pre-packed columns (contain 1mL nickel-chelate agarose), prepared wash and elution buffers make set-up and execution very easy
Convenient size – five columns and sufficient reagents for five purifications
Product Details:
Purification of histidine-tagged protein from S. cerevisiae using the Y-PER 6xHis Fusion Protein Column Purification Kit. Strain DY150 carrying plasmid pYIL042C was grown and induced with galactose to produce a histidine-tagged protein. Induced cells were harvested at OD600 = 5.3 and lysed using Y-PER Reagent for 20 minutes. Extract was purified following the default kit procedure. Fractions were collected from each of the purification steps and assayed by 4%-20% SDS-PAGE. The gel was stained with GelCode Blue Stain Reagent (Part No. 24590). Lane 1: crude lysate extracted from E. coli expressing His-tagged green fluorescent protein using B-PER Reagent; Lane 2: flow-through of the lysate after loading onto a Ni-chelated column; Lanes 3-4: two washes with 3mL of 6xHis Wash Buffer 1; Lanes 5-7: three washes with 3mL of 6xHis Wash Buffer 2; and Lanes 8-9: 6xHis-tagged GFP eluted from the column with 6xHis Elution Buffer. Lane M: MW markers.
Thermo Scientific Y-PER 6xHis Fusion Protein Column Purification Kit uses Y-PER Yeast Protein Extraction Reagent and nickel-IDA (Ni-IDA) agarose columns to extract yeast cells and purify His-tagged fusion proteins.
Purification of histidine-tagged protein from S. cerevisiae using the Y-PER 6xHis Fusion Protein Column Purification Kit. Strain DY150 carrying plasmid pYIL042C was grown and induced with galactose to produce a histidine-tagged protein. Induced cells were harvested at OD600 = 5.3 and lysed using Y-PER Reagent for 20 minutes. Extract was purified following the default kit procedure. Fractions were collected from each of the purification steps and assayed by 4%-20% SDS-PAGE. The gel was stained with GelCode Blue Stain Reagent (Part No. 24590). Lane 1: crude lysate extracted from E. coli expressing His-tagged green fluorescent protein using B-PER Reagent; Lane 2: flow-through of the lysate after loading onto a Ni-chelated column; Lanes 3-4: two washes with 3mL of 6xHis Wash Buffer 1; Lanes 5-7: three washes with 3mL of 6xHis Wash Buffer 2; and Lanes 8-9: 6xHis-tagged GFP eluted from the column with 6xHis Elution Buffer. Lane M: MW markers.
