JoVE Encyclopedia of Experiments
Microbiology
0 views • 3:45 min • August 31st, 2026
Begin with a suspension of bacterial cells expressing polyhistidine-tagged viral integrase.
Sonicate the suspension to disrupt the bacterial membranes and release the integrase.
Transfer the lysate to an ultracentrifuge tube.
Centrifuge at high speed to pellet the cell debris and collect the supernatant.
Next, take a chromatography column packed with nickel-charged resin and equilibrate it with buffer.
Load the clarified lysate onto the column.
Proteins with histidines bind weakly to nickel, while polyhistidine-tagged integrase binds strongly.
Wash with buffer to remove unbound proteins, then add elution buffer with a linearly increasing imidazole concentration.
Imidazole competes with histidine for nickel binding and releases the integrase.
Collect the fractions and transfer aliquots to tubes containing loading buffer.
Heat to denature the proteins, then load onto a polyacrylamide gel.
Perform electrophoresis and analyze the protein bands.
Identify fractions containing pure integrase, without any contaminating proteins, and pool them to obtain purified viral integrase.
Begin by thawing one pellet of E.coli expressing prototype foamy virus integrase on ice. Use a sonicator equipped with a 0.5-i
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