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LOV2 Purefication From Raw Cell Extract

Back to Results

Aims

Methods

Preparation of the calmodulin affinity resin column A calmodulin affinity resin column was set up with a retort stand and partly filled with binding buffer. 2ml of CAM affinity buffer plus a little more binding buffer were added to the column and allowed to flow through to form a packed bed, with the flow through collecting in a glass beaker underneath. Finally the resin was washed with 10ml more of binding buffer, causing as little disturbance to the resin bed as possible. This was to prepare the column for the addition of an E.coli total protein extract containing the PHOT1 LOV2 domain fused to a CBP tag .

Affinity purification of LOV domain Of a 2ml centrifuged sample of protein extract, 1.8ml was loaded into the column (leaving the remaining 0.2ml for SDS-PAGE analysis) and allowed to flow through and collect into a microfuge tube. The liquid in the column turned yellow after this addition as the protein passed through the tube. The column was then subsequently washed with 3ml of binding buffer and any flow through collected in the same tube. The LOV domain was eluted by pipetting 0.5ml of elution buffer into the column and collecting the eluate in a microfuge tube before immediately placing on ice. This was repeated six more times to give a total of seven different labelled eluates, each with different concentrations of LOV domain in them.

Preparation of samples for SDS-PAGE

With the eluates cooling on ice, 12.5µl of the remaining column load, the flow-through fraction and each eluate were mixed with 12.5µl of 2xSDS loading buffer (giving a final volume of 25µl). This was then immediately heated to 100°C to prevent denatured proteins being destroyed by any protease present, then allowed to cool.

Measurement of protein concentration A Bradford assay was then carried out to determine the concentration of LOV protein in the eluates using a sample of each eluate diluted to 1ml with water, 0.9ml Bradford reagent and 0.1mg/ml BSA stock solution, the volume of which was calculated so that the standard curve would cover 0.5-10µg of BSA. A control containing no protein was also included in the standard curve for comparison. Each sample was then mixed and placed in a plastic cuvette and the absorbace of each measured in a spectrophotometer at 595nm. The absorption values of the column load, flow through and eluates were also measured by making a 10-fold dilution of the column load and flow through, then transferring 10µl of these, plus 10µl of each eluate to separate microfuge tubes (in duplicate). 90µl of binding buffer and 900µl of Bradford reagent were also added to each tube before decanting into a plastic cuvette and measuring the absorption in the same way as before. The concentration of LOV protein in each sample was calculated using the standard curve drawn from the BSA absorptions.

Afterwards the samples were illuminated with a UV lamp in a darkroom. Any LOV protein present fluoresced yellow as expected, even though it appeared colourless under normal light conditions. After inspection the samples were placed back on ice to remain cool until an SDS-PAGE gel had been prepared.

Preparation of SDS-polyacrylamide gel

The 12% separating gel was set up by mixing the following ingredients in a conical flask (Table 1):

Table 1: components of 12% separating gel used for SDS-PAGE analysis of LOV protein purity

As soon as these components were mixed the gel was poured, then overlayed with water-saturated butanol and left to set. A 5% stacking gel was then prepared in a similar way to the separating gel, this time using the following volumes (Table 2): Table 2: components of 5% stacking gel used for SDS-PAGE analysis of protein purity

Once the separating gel had set, the water-saturated butanol was removed and the newly prepared stacking gel added in its place. A plastic comb was placed into the gel to create loading wells for the samples to be pipetted into once fully prepared. On solidifying, the comb was removed from the stacking gel and the gel tank was assembled. The tank was then filled with SDS running buffer, and 25µl of each of the purified, defrosted samples was added into each well. Into a final well was added 5µl of pre-stained molecular weight markers (Table 5), then, once connected to the power supply, the gels were run at 80 volts for approximately one hour, until the samples had nearly reached the bottom of the gel.

After the gel had run for the appropriate length of time, the apparatus was dismantled and the gel was placed in a box, ready to be stained.

Staining gels for protein Enough coomassie blue stain was added to cover the gel, then was left for roughly 15 minutes. Water was then used to rinse any excess stain from the gel, keeping the stain for re-use later.

Scanning and analysis

The gel was then carefully placed between 2 sheets of acetate and scanned. The scan was then analysed to see which lanes contained the highest concentration of LOV protein, and which lane had the purest sample. A graph of mobility against log Mr was drawn to estimate the Mr of LOV protein.

Results

LOV domain purification 15 samples containing BSA were made up for the Bradford assay for the purification of LOV domain protein, ranging from 0mg/ml to 10mg/ml. The volume of BSA in each sample was then calculated so that the appropriate volume of water could be added to ensure each sample was the same volume in total (0.1ml). The absorption of each sample were recorded (Table 3) then used to plot a standard curve (Figure 1).

Table 3: BSA concentration and volume used in each sample for the Bradford assay, and absorption of each sample used to create a standard curve

BSA concentration (mg/ml) BSA volume (µl) Absorption of light (at 595nm) 0.0 0 0.000 1.0 10 0.008 1.5 15 0.025 2.0 20 0.029 2.5 25 0.032 3.0 30 0.043 4.0 40 0.074 5.0 50 0.078 6.0 60 0.091 7.0 70 0.114 8.0 80 0.127 9.0 90 0.132 10.0 100 0.145

The absorption values measured show an increase in value as the concentration of BSA in each sample increases, giving a fairly straight line on the standard curve. The absorption values recorded for the 7 different eluates, the flow through and the column load were carried out in duplicate with average values calculated for any values which appeared largely deviant from each other in each sample. The concentration of each sample was then read from the standard curve by using these absorption values (Table 4).

Figure 1: Standard curve of BSA concentration in each sample against absorption of light at 595nm to calculate the concentration of LOV domain protein in each of the prepared eluates.

Table 4: Absorption of 595nm light of each sample of purified LOV protein including column load and flow through, plus concentration of each sample calculated from the standard curve.

Sample Absorption of light (at 595 nm) Concentration (µg/ml) Column load 0.304 10.6 Flow through 0.186 2.8 0 0.000 0.0 1 0.169 1.8 2 0.226 5.4 3 0.246 6.9 4 0.172 11.9 5 0.174 2.0 6 0.151 0.7 7 0.147 0.3 When placed under the UV lamp, samples 4, 3 and the column load showed strongest fluorescence as these had the highest concentration of LOV protein present. 3.2. Determination of LOV domain protein purity Figure 2: SDS-PAGE gel stained with coomassie blue showing the varying concentrations of LOV protein in each sample. FT – flow through; CL – column load; 1,2,3,4,5,6,7 – samples 1 to 7; MM – molecular markers. On scanning the SDS-PAGE gel with coomassie blue stain, the 9 different lanes could be clearly identified. The flow through and column load lanes show multiple bands of proteins as these were the impure samples. The column load has dark bands as there was a large concentration of protein present. The molecular markers also appear clearly. Of the purified samples, lane 3 shows the darkest band as this lane contains the highest concentration of protein. Lane 3 also contains a relatively pure sample as there are very few background bands of protein. Lane 4 however has no bands present other than the LOV protein, so this sample was one of the purest of the 7.

Using the distance travelled by the molecular markers and their weights (Table 5), a graph of mobility against log Mr was plotted to estimate the Mr of LOV protein (Figure 3). Table 5: Weight of molecular markers used in SDS-PAGE plus distance migrated down the gel. Figure 3: Standard curve of the distance travelled by each molecular marker protein down the SDS-PAGE gel against log Mr to calculate the molecular weight of LOV in the protein sample. The LOV protein bands were measured to have travelled 32mm down the gel. From the graph it was thus calculated that the LOV protein weighed 18,000 Daltons so would appear between lysozyme and β-lactoglobulin on the graph. 3.3 Flavin excitation and content 3.3.1. Spectroscopy The scans of the LOV protein after being left in the dark, then irradiated for 1 minute, then darkened again appeared as shown below (Figure 4). FMN causes the three peaks seen on the scan, and the absorption seen at the beginning of the scan is caused by aromatic sidechains on the protein. 3.3.2. Binding stoichiometry The FMN solution concentrations and their absorption of 450nm light in the spectrophotometer are shown below (Figure 5).

Table 5: Concentrations of FMN solution made between 2µM and 20µM, and the absorption of each solution in a spectrophotometer at 450nm needed to create a standard curve. FMN concentration (µM) Light absorption (at 450nm) 2 0.027 7 0.098 11 0.133 15 0.180 20 0.234 These values were then plotted to create a standard curve. (Figure 4). The absorption of the supernatant after boiling was also measured at the same wavelength and recorded to have a value of 0.085. Using this value, the concentration of supernatant in the sample was read from the graph to be 7.1µM Using the results gathered from experimentation up to this point, the stoichiometry of FMN binding to the LOV domain was calculated. FMN concentration of supernatant = 7.1µM

Concentration of eluate fraction 3 (eluate with highest concentration) = 6.9µg/ml

Concentration of eluate fraction 2 (eluate with second highest concentration) = 5.5µg/ml

However the protein sample used for the Bradford assay was diluted 100 fold thus the concentrations used for eluate 2 and 3 are 100 times too small, therefore;

Concentration of eluate fraction 3 = 690µg/ml = 0.69mg/ml

Concentration of eluate fraction 2 = 540µg/ml = 0.54mg/ml

Average concentration = 0.62mg/ml = 0.0062g/ml

Estimate of Mr for LOV protein = 18,000 Da

= 18 g/ml 1M

= 18/1000 g/ml 1mM

Concentration of LOV = 0.0062 / 18 = 27.8µM

Amount of FMN in sample = 7.1/27.8 x 100 = 25.5%