How pure is the elution sample compared to the control

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Reference no: EM1397479

You are asked to run an SDS-PAGE analysis of samples from an affinity chromatography experiment

performed by your TA. A recombinant protein with a hexahistidine tag was captured in a nickel- agarose bead matrix and eluted off the column with 75mM imidazole. Your TA was hoping to make about 2 mg/mL of the protein. You are given the following samples to prepare and load into an SDS-PAGE gel :

· 10 uL 75 mM Imidazole

· 10 uL pure recombinant protein at 2 mg/mL

· 10 uL recombinant protein in 75 mM imidazole (elution fraction)

1. Describe how you would prepare the three samples to load into the gel.

2. Label the gel lanes with the appropriate contents. The recombinant protein should exhibit one band at 66,000 Daltons

3. Compare the elution lane to the positive control lane. How do you know the recombinant protein present in the elution sample? How pure is the elution sample compared to the control?

4. What assumption can you make about the amount of recombinant protein in the elution sample? What assay can you perform to find the accurate concentration of the elution sample?

Reference no: EM1397479

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