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problem with Enzyme purification


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#1 Dravid

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Posted 18 January 2013 - 03:30 AM

Well. I am trying to purifiy an enzyme using blue agarose affinity beads. The enzymatic activity in the crud extract is 45 units/ml. When i incubated 2ml of crude extract with 5 ml of packed volume of beads and took out 0 Hrs fraction immidiately the activity became 20 units/ml. Now i am trying to elute the enzyme from the beads, but it is not working even using 10 mM of NADH as an elution buffer. I couldn't figure it out where is the enzyme has gone. Is it degraded or tightly bound to the beads?

#2 mdfenko

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Posted 18 January 2013 - 05:01 AM

probably tightly bound. what are the complete conditions (eg salt concentration, buffer, pH, other cofactors...)?
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#3 Dravid

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Posted 18 January 2013 - 09:02 AM

Well initially the beads were equilliberated using 50mM Tris.HCl buffer(pH 6.8) and before incubated with the beads, the crude extract was diluted two times using 50 mM Tris HCl(pH6.8). For washing again the same buffer was used and finally was trying to elute the enzyme using 1 to 10mM of NADH prepared in 50mM Tris HCL(pH6.8).

#4 Dravid

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Posted 18 January 2013 - 09:03 AM

probably tightly bound. what are the complete conditions (eg salt concentration, buffer, pH, other cofactors...)?

Well initially the beads were equilliberated using 50mM Tris.HCl buffer(pH 6.8) and before incubated with the beads, the crude extract was diluted two times using 50 mM Tris HCl(pH6.8). For washing again the same buffer was used and finally was trying to elute the enzyme using 1 to 10mM of NADH prepared in 50mM Tris HCL(pH6.8).

#5 mdfenko

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Posted 18 January 2013 - 02:43 PM

i've found that blue agarose works better in the presence of salt (i've routinely used 0.5M nacl in the loading and elution buffers). you may want to use at least 0.1M (for starters).

from the booklet "affinity chromatography" (ge healthcare):
"For elution use low concentrations of the free cofactor, NAD+ or NADP+ (1–20 mM), or increase ionic strength (up to 2 M NaCl or KCl, 1 M is usually sufficient).
For less specifically bound proteins: use higher concentrations of cofactor or salt or more severe eluents such as urea or potassium isothiocyanate. Polarity reducing agents such as dioxane (up to 10%) or ethylene glycol (up to 50%) may be used."


you can download the handbook here (the pdf download should start when you enter the page): affinity chromatography: principles and methods.

talent does what it can
genius does what it must
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