Completion of ADH Purification using Affinity Column Chromatography...

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Completion of ADH Purification using Affinity Column Chromatography and Concentrating
ADH using Ammonium Sulfate Precipitation:
When performing protein purifications, it is necessary to take detailed notes on each preparation. Include any protocol variations (intentional or not) or any problems It is also important to save samples at each step along the way for analysis When developing a protein purification strategy for a new target protein, it is critical to evaluate each step for advantages in purification relative to disadvantages in overall yield This requires careful analysis of samples from each step with respect to volume. enzyme activity, protein concentration and protein composition. Even when performing establish protocols for protein purification it is necessary to save samples along the way to ultimately
check the efficiency of the purification procedure. Keeping track of protein yields during purification (cell pellet mass, volumes and enzyme concentrations at each step from crude extract through final pooled fractions) will facilitate troubleshooting if problems arise. In general it is a good practice to 'save everything' until you have experimental confirmation that the protein preparation was a success.
Many biochemical procedures require specific buffers. Some reagents necessary for one experiment may be problematic for subsequent experiments. Thus, it is often necessary to exchange the buffer of a protein sample between experiments. The buffer solution of a protein sample can be exchanged using a technique called dialysis. This relies on a porous cellulose membrane that limits diffusion based on size. Large molecules like proteins can be retained within the dialysis tubing while smaller molecules, reagents and ions can pass through the pores. To exchange the buffer of a protein solution, it is placed within dialysis tubing and the dialysis tubing is placed within a larger volume of the desired buffer solution (dialysate) with gentle stirring. It may take several buffer changes over many hours to ensure
complete buffer exchange.
It is often necessary to concentrate protein samples during protein purification and this can be done using ammonium sulfate precipitation. In this technique, the protein solution is brought to a high concentration of ammonium sulfate, ions which are highly soluble in aqueous solutions. As the ammonium sulfate concentration in the solution is increased. the water molecules (which would otherwise be interacting with any charged or polar surface residues of the proteins) interact with the ammonium and sulfate ions. Since the proteins are no longer interacting with the water molecules. they are formed to interact with each other and precipitate out of solution. All proteins precipitate out of
solution once the ammonium sulfate concentration rises above -55%. Using an ammonium sulfate concentration beyond this value will precipitate all proteins within the sample A pure protein pellet can be obtained upon centrifugation and the proteins can be resuspended in any volume of a desired buffer.
Note that a fair amount of residual ammonium sulfate is still present in this pellet
Ammonium sulfate precipitation can also be used as a purification step in its own right. A protein's physical properties determine the concentration of ammonium sulfate at which it precipitates out of solution. Thus, if a target protein can remain in solution at a higher concentration of ammonium sulfate compared to other contaminating proteins in the sample, this difference can be exploited to purify the protein. If that solution is brought to an ammonium sulfate concentration just below that required to precipitate the target protein, then the other contaminating proteins can be removed from the solution
Alternatively, if the target protein precipitates out of solution at a much lower ammonium sulfate concentration than the other contaminating proteins in the mixture then the target protein can be selectively precipitated out of solution away from the contaminants.
4. What is the purpose of the 2nd wash step?
5. What is the purpose of the dialysis step after purification on the nickel column?
6. What ammonium sulfate concentration (final concentration) will be used to precipitate the other half of your purified ADH?

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4. Purpose of a second wash is to further remove any loosly binded proteins. 2nd wash buffer contains 100 mM imidazole and it should...

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