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Purification Table Instruction

The document provides instructions for creating a purification table to present data from protein purification steps. The table requires the volume, total protein, activity units, total activity, specific activity, fold purification, and percent yield for each fraction. Specific activity is a measure of purity, calculated by dividing activity by total protein. Fold purification compares the specific activity of each fraction to the starting lysate. Percent yield indicates the amount of activity recovered from each step. An example purification table is given to demonstrate the calculations.

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17K views

Purification Table Instruction

The document provides instructions for creating a purification table to present data from protein purification steps. The table requires the volume, total protein, activity units, total activity, specific activity, fold purification, and percent yield for each fraction. Specific activity is a measure of purity, calculated by dividing activity by total protein. Fold purification compares the specific activity of each fraction to the starting lysate. Percent yield indicates the amount of activity recovered from each step. An example purification table is given to demonstrate the calculations.

Uploaded by

api-3744715
Copyright
© Attribution Non-Commercial (BY-NC)
Available Formats
Download as PDF, TXT or read online on Scribd
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Purification Table

Instruction
MSUM Biochemistry

Theory and Introduction: A purification table is used after a series of separation steps. This table, similar to
the one you observed in the purification tutorial, is used to present the information about the yield and purity of
the purification. There are only a couple of data needed to prepare the table:
• Volume of each step of the purification, starting from the lysates, to the final pooled sample.
• Total protein (mg) for each step.
• Units (amount) of protein being purified (typically done using the enzyme activity units).
The rest of the information for the table can be calculated from this data.

Example Purification Table


Volume Total Protein Activity Total Activity Specific Activity Fold
Fraction (ml) (mg) (units) (units * ml) (units / mg) Purification % Yield
Lysate 10.00 56.3 65897 658970 1170 1.0 100
DEAE 12.50 25.36 42845 535563 1689 1.4 81
Ni-Agarose 8.25 18.25 51481 424718 2820 2.4 64
S-200 4.30 3.75 48239 207428 12863 11.0 32
Reactive Blue 3.50 1.28 46578 163023 36389 31.1 25

Calculations –
1. Units – Normally, this is determined using an enzymatic assay. MGH has an enzyme, as one of the fusion
partners, but we are determining the fluorescence of the protein as a function of the enzyme. THIS
MUST BE DONE AT THE SAME TIME. You must thaw the fraction of your lysate, pooled samples and
final purification sample and conduct a fluorescent assay at the same time. Different settings on the
plate reader will give you a different result, thus it must be read at the same time using the same plate.
Simply thaw the samples and place 100 µl into a well. Return the samples back to the tube for later assay.
The units of activity will thus be measured as MGH Fluorescence (relative fluorescent units). For
specifics on how to determine enzymatic activity, go to the lab website for that handout.
2. Total Activity – this is a measure of how many units of protein you have in a sample. Simply multiply the
activity in the sample by the total volume.
3. Specific Activity – specific activity is a way to measure how much of a measured protein (MGH in this
case) there is with all of the other contaminating proteins. Divide the Activity by the mg of protein. The
higher this value, the higher purity.
4. Fold Purification – Divide the specific activity of each fraction by the specific activity found in the
lysates. This number changes depending on the protein you are working with. There is no good or bad
value. However, this number along with the percent yield, indicates if a step was worthwhile or not. A
poor fold purification with a low yield is a step to avoid in the future, while a high fold purification and
high yield is a great thing…
5. Percent Yield – Calculate the percentage of the yield for each step using the total activity from the
starting step.

Try setting up an excel spreadsheet using the formulas given above.

1 October 06

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