Uniform Films

Background

New proteins were produced adhering rigorously to the protocol laid out in the His-tag purification protocol with all buffers pH checked prior to use. The results of the SDS-PAGE shows large protein yields and the greatest source of loss in the second denaturing washing step which suggests the greater variability of the buffer at this pH compared to 6.0. This is further compounded when considering the protocol suggests the preparation of this buffer from taking an aliquot from the pH 6.0 buffer; small volumes lead to larger changes in pH.

The resulting proteins were concentrated by acetone precipitation.

The main focus is to achieve uniform colouration.

Outline Plan

The main focus of this experiment was to attempt to achieve a uniform film of one or two colours. It is proposed to tackle this with the knowledge gained from our previous breakthrough:

• Dilute samples in less HFIP (50μl) to achieve greater protein concentration relative to solvent
• Spin with less volume (2μl) using approximately 1.5cm x 1.5cm piranha cleaned silicon substrate
• Attempt to strike a balance between solvent evaporation and spreading by adjusting spin speed and time

In addition samples from both the newly acetone precipitated reflectins and previous reflectins obtained from dialysis were spun to guage the resulting quality.

Results and Observations

From the outset the results were remarkable. The acetone precipitated proteins yielded a brilliant uniform blue colour which it retained for some time prior to crystallisation. By lowering the spin speed a two tone 'gradient' film of yellow and blue was obtained. A subsequent drop cast of the sample indicated yellow was the longest wavelength achievable. However films produced eventually suffered crystallisation, interestingly it was noted the onset of crystallisation became more prominent the more you used of the same sample which somewhat indicates in the sample preparation, centrifugation somewhat fractionates the protein and urea with less urea in the upper layers compared to the lower layers.

Similarly the dialyzed proteins yielded similar levels of uniformity. A different problem however presented themselves here. Whilst they did not tend to crystallise, the films produced tended to lose their colouration much faster and solvent evaporation was much slower. These effects became more prominent as one worked their way through the sample which again suggests fractionation. It is hypothesized that this phenomenon is due to the contaminating tris salt which is deposited regularly on the silicon wafer and impedes solvent evaporation and promotes de-wetting of the films. Filtering with a 0.22μm filter tip seems to help however this is only feasible given the available equipment if large volumes are available and much of the solution is lost in the syringe and filter after filtration making it infeasible in practice to implement.