Recombinant IRF5 dimerization assay - protein preparation

IRF5 PURIFICATION PROTOCOLS

Cell Pellet Preparation: All cells are induced at 23°C with 0.5mM IPTG overnight (50µM Biotin is added to constructs with Avitags). Cells are harvest via centrifugation, washed once in PBS and stored at -80°C until purification. 

IRF5 (222-467) S430D-Histag 

Lysis Buffer: 500mM NaCl, 50mM HEPES pH 8.0, 10% glycerol, 1mM DTT

HisTrap Buffer A: 500mM NaCl, 50mM HEPES pH 8.0, 10% glycerol, 1mM DTT

HisTrap Buffer B: 500mM NaCl, 50mM HEPES pH 8.0, 10% glycerol, 1M imidazole, 1mM DTT

Sizing Column Buffer: 300mM NaCl, 20mM HEPES pH 8.0, 10% glycerol, 1mM DTT

PROTOCOL (All steps carried out at 4°C unless otherwise noted)

1. The cell pellet from a 6L E.Coli TB prep is re-suspended in Lysis Buffer with 20U/mL benzonase and complete Roche Protease Inhibiters, EDTA free (Cat No. 05056489001).
   a. 1g of cell per 8mL of Lysis  Buffer
2. Cell suspension is passed through microfluidizer, 3Xs.
3. The lysed cell suspension is centrifuged at 14,000rpm in Sorvall 1500 rotor for 45min.
4. The supernatant is separated from insoluble cellular debris and passed through 0.22µm filter followed by the addition of 20mM imidazole.
5. The filtered supernatant is loaded onto 2x5mL GE Healthcare HisTrap HP columns (Cat No. 17-5247-01), equilibrated with 98% Histrap Buffer A and 2% HisTrap Buffer B, at 5mL/min followed by ≥10CV wash with 98% Histrap Buffer A and 2% HisTrap Buffer B, at 5mL/min or until Abs280 stabilizes.
6. The HisTrap HP columns are eluted with 2-50% HisTrap Buffer B gradient over 20CV with a flow rate of 5mL/min and 4mL fraction collection.
   .       Fractions are analyzed using SDS-PAGE. Fractions containing target protein are pooled.
7. The pooled fractions are concentrated to an appropriate volume for loading onto a HiLoad Superdex 75 26/60 prep grade (Cat No. 17-1070-01) using 10kDa NWCO Amicon Centrifugal Filter Device (Millipore Cat No.UFC9010).
8. The concentrated sample is loaded onto a Hiload Superdex 75 26/60 prep grade, pre-equilibrated with Sizing Column Buffer, with a running flow rate of 2.5mL/min and 4mL fractions after 0.3CV.
   .       Fractions are analyzed using SDS-PAGE. Fractions containing target protein are pooled.
9. The pooled fractions are concentrated to desired concentration using 10kDa NWCO Amicon Centrifugal Filter Device (Millipore Cat No.UFC9010).    
   .       Final concentration is measured using Absorbance at 280nm.

IRF5 (222-467) S430D-AviTag

Lysis Buffer: 500mM NaCl, 50mM HEPES pH 8.0, 10% glycerol, 1mM DTT 

Monomeric Avidin Column Buffer A: 500mM NaCl, 50mM HEPES pH 8.0, 10% glycerol

Monomeric Avidin Column Buffer B: 500mM NaCl, 50mM HEPES pH 8.0, 10% glycerol, 2mM d-Biotin

Dialysis Buffer/Sizing Column Buffer:  100mM NaCl, 20mM HEPES pH 8.0, 10% glycerol, 1mM DTT

PROTOCOL (All steps carried out at 4°C unless otherwise noted)

1. The cell pellet from a 6L E.Coli is re-suspended in Lysis Buffer with 20U/mL benzonase and cmplete Roche Protease Inhibiters, EDTA free (Cat No. 05056489001).
   a. 1g of cell per 8mL of Lysis Buffer
2. Cell suspension is passed through microfluidizer, 3Xs.
3. The lysed cell suspension is centrifuged at 14,000rpm in Sorvall 1500 rotor for 45min.
4. The supernatant is separated from insoluble cellular debris and passed through 0.22µm filter.
5. The filtered supernatant is loaded onto 20mL Pierce ®Monomeric Avidin Agarose (Cat No. 20267) Column (irreversible sites blocked with 2mM d-Biotin as per manufacturer’s instructions and equilibrated with Monomeric Avidin Column Buffer A) at 0.5mL/min overnight.
6. After supernatant was loaded, the Monomeric Avidin Agarose column was washed with ≥5CV of Monomeric Avidin Column Buffer A at 2mL/min.
7. The column was eluted with Monomeric Avidin Column Buffer B with manual fractions of 4mL each at 2mL/min flow rate
   . Fractions are analyzed using SDS-PAGE. Fractions containing target protein are pooled.
8. The pooled fractions are concentrated to an appropriate volume for loading onto a Hiload Superdex 75 26/60 prep grade (Cat No. 17-1070-01) using 10kDa NWCO Amicon Centrifugal Filter Device (Millipore Cat No.UFC9010).
9. The concentrated sample is loaded onto a Hiload Superdex 75 26/60 prep grade, pre-equilibrated with Sizing Column Buffer, with a running flow rate of 2.5mL/min and 4mL fractions after 0.3CV.
   .   Fractions are analyzed using SDS-PAGE. Fractions containing target protein are pooled.
10. The pooled fractions are concentrated to desired concentration using 10kDa NWCO Amicon Centrifugal Filter Device (Millipore Cat No.UFC9010).  
    .   Final concentration is measured using Absorbance at 280nm.

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