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Gapdh Lab Report

Decent Essays
Abstract

Affinity purification is a powerful method of isolating a protein of interest from a complex mixture of proteins. the aim of this experiment was to isolate the enzyme GAPDH from a complex mixture of proteins, in this case yeast lysate. in order to achieve this, a range of methods were used such as protein, GAPDH activity and ADH assays, SDS-PAGE and imumunodetection of ADH and GAPDH on western blot. The results showed that the isolation of GAPDH was achieved albeit, extremely with the % recoveries of the elution being abnormally high due to abnormally low initial material values. The results suggested overall that isolation GAPDH from yeast lysate using affinity purification is in fact a somewhat effective method.

Methods

Affinity
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SDS-PAGE Electrophoresis Gel. - Expression of numerous proteins in different fractions of yeast lysate. Marker proteins were utilised in order to identify them under reducing conditions. Polyacrylamide gel was stained with Instant Blue Coomasie Stain which binds itself to proteins allowing band patterns to be viewed. 10ul of molecular weight standards was expelled in the first lane with the remaining fractions in the following lanes. GAPDH rabbit muscle and Yeast ADH was used as a control.
Figure 2. Immunodetection of GAPDH on Western Blot. - Expression of GAPDH in yeast lysate fractions. Presence of GAPDH was assessed using its antibodies. Proteins were transferred from the SDS-PAGE gel to a nitrocellulose membrane for the proteins to be readily bound to the antibodies using an electric current. The membrane was then incubated with the antibodies with the enzyme alkaline phosphate which was used to indicate the presence of GAPDH

Discussion

The aim of the experiment was to isolate GAPDH, from table 2 it
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It was especially high in elution 1 and 2 who had values of 527% and 514% respectively, indicating a high GAPDH activity especially elution 1. This is supported by the fact that elution 1 had the highest specific activity with a value of 0.0017 umoles/min/mg. it is however difficult to see in figure 2, with the number of bands being seemingly lower than there should be.

The SDS-PAGE Electrophoresis (figure 1) contained different samples of the yeast lysate. From the gel the bands are clear and distinct with the YNSMY-YNFT having the thickest and most distinct of columns making the cibracon blue an efficient tool for affinity chromatography. The protein concentration from YNSM to YNW4 fluctuated with a general decreasing trend however the gel does not distinctively show this.

Limitations

The % protein recovery from the ADH assay was 556%, which could be due to a dilution error, most likely that the initial material had abnormally low values. It was expected that elution 3 (YNE3) would have the lowest activity in GAPDH, from table 2 it is clear that it had the highest specific activity but the lowest total activity and thus the lowest % protein recovery.
Overall, the results suggest that, this was somewhat of an effective method in isolation the enzyme GAPDH from yeast
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