MJohnson MMBB380 Report 4-5

8/2/2019 MJohnson MMBB380 Report 4-5

1/5

Mark Johnson

MMBB 382-53

Exp. 4/5(9-22-10/9-29-10)

10-6-10

- Ion Exchange Chromatography / Bradford Assay and SDS PAGE

Data:

0

0.1

0.2

0.3

0.4

0.5

0.6

0 50 100 150 200 250

Time (sec)

Abs@340nm

FIGURE 1.

Activity of Isocitrate Dehydrogenase (IDH) in various samples ofChlamydomonas

The originating sample was derived from a vortexed and centrifuged aliquot ofChlamydomonas cells. To the supernatant, ammonium sulfate, (NH4)SO4, was then added

to a concentration of 35%. This solution was centrifuged, and the supernatant was

collected. The solution was dialyzed to remove as much (NH4)SO4 as possible. The

dialyzed solution was then eluted through Q ion exchange chromatography column (wherethe matrix was Q Sepharose with the functional group quarternary ammonium) using

increased concentrations of potassium chloride, KCl. The black line represents the

dialyzed 35% supernatant solution before elution. The pink line represents the flow-thru(the first allotment of sample extracted from the column after adding Tris buffer). The

yellow line represents the wash - the rest of the allotment of Tris buffer added drained

from the column before adding KCl. The light blue line represents the sample collectedafter adding 40 mM KCl to the column. The purple line represents the sample collected

after 60 mM KCl was added to the column. The dark blue line represents solution

collected after adding 80 mM KCl to the column. The green line represents the sample

collected after 100 mM KCl was added to the column. The orange line represents thesample collected after 200 mM was eluted through the column. Each of the samples were

measured by a spectrophotometer at 340nm for 400 seconds, and absorbance was recorded

every 15 seconds.


2/5

TABLE 1

Elution of a sample ofChlamydomonas cells through a Q ion exchange

chromatography column (measuring IDH)

A dialyzed sample of 35% (NH4)SO4 was eluted through a Q ion exchange chromatography

column using increasing concentrations of KCl in Tris buffer to attempt to purify the IDHfrom the sample. 2.0 mL of each KCl solution was added to the column and the eluate was

collected. The samples collected from each increase in KCl were assayed to determine the

activity of IDH using isocitrate. Total protein concentration was determined by performinga Bradford assay of each sample.

_______________________________________________________________________

Sample Sample Total Activitya % Original Total SpecificType Volume Volume IDH Activityb Protein Activity

Concentrationc

________________________________________________________________________

L mL M/min % g/L nmol/ming

35% S 25 4.3 7.58 100 3.286 0.1845

Flow-thru 80 3.9 12.42 46 1.704 0.1822

Wash 80 3.3 7.58 24 0.2966 0.6390

40 mM KCl 80 2.2 4.19 9 0 0

60 mM KCl 80 2.6 2.58 6 0.01610 4.0070

80 mM KCl 80 2.6 3.06 8 0 0

100 mM KCl 80 2.3 2.42 5 0 0

200 mM KCl 80 2.5 0.65 2 0.7502 0.02166

________________________________________________________________________

a Data generated using information from Figure 1 from time points 180 seconds to 240seconds. The Beer-Lambert Law was used to determine the values.

b Percentages all based on dialyzed 35% sample ofChlamydomonas its activity

designated 100%c Trendline equation from graph of BSA standards used with data from Bradford assays to

determine values y = 0.0205x + 0.0874


3/5

FIGURE 2.

SDS PAGE gel of various samples ofChlamydomonas

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS PAGE) was used toanalyze the protein in the samples. The samples used were derived from the elution of a

sample ofChlamydomonas through an ion exchange column (as described above). Each

lane contained a different sample. The first lane (left to right) contained a broad rangeprotein standard, the weights of each protein in the standard (each band) is known shown

to left of picture. The second lane contained the 35% supernatant sample. The third lane

contained the flow-thru sample. The wash sample was placed in lane 4. In lane 5, a

sample of 40 mM KCl eluate was placed. Lane 6 contained the 60 mM KCl eluate sample.The seventh lane held the 80 mM KCl eluate sample. Lane 8 contained the 100 mM KCl

eluate sample. The ninth lane contained the 200 mM KCl sample. A 5-20% gradient gel

was used. The gel was run for one hour and 150 volts.

209,000 kDa

123,000 kDa

80,000 kDa49,100 kDa

34,800 kDa28,900 kDa

20, 600 kDa

7,100 kDa


4/5

Results:

In Figure 1, various samples containing Chlamydomonas extract are assayed forabsorbance of IDH (the samples are explained in the legend below Fig. 1). Each line

shows the relationship of time versus the absorbance of the corresponding sample. The

time span of the procedure was 240 seconds. The flow-thru sample achieved the highestabsorbance value of 0.477. The flow-thru line seems to have the steepest slope. The

lines representing the 40mM KCl, 60 mM KCl, 80 mM KCl, 100 mM KCl, and the 200

mM KCl samples have almost flat slopes. The 35% S sample started with the lowestabsorbance value of all the samples ,0.08, and had a final absorbance of 0.284, which was

the lowest final absorbance of any of the samples. All of the lines show a positive

relationship between time and absorbance, and they are all relatively linear.

Table 1 shows the initial volume of the Chlamydomonas sample, which is 4.3 mL. The

other solutions were of various volumes, since they are samples of eluate collected from

the ion exchange column. The lowest volume was the 40 mM KCl sample at 2.2 mL. The

35% S solution contained 100% of the IDH activity. The percentage decreased with eachsuccessive sample, the lowest being the 200 mM KCl sample with 2% of the original IDH

activity. The flow-thru sample displayed the highest activity of 12.42 M/min. Thelowest activity value, 0.65 M/min , was received from the 200 mM KCl sample. The 35%

S solution had the most total protein concentration of 3.286 g/L. The 40 mM KCl, 80

mM KCl, and the 100 mM KCl samples all returned a value of zero for the total protein

concentration. These same samples also produced a specific activity value of zero. The 60mM KCl solution presented the highest specific activity, whereas the 200 mM KCl solution

presented the lowest non-zero specific activity.

Figure 2 shows the SDS PAGE gel in which each of the Chlamydomonas extract samples

used to create Fig. 1 was run. Each lane contains a different sample, explained in the

legend. The first lane (farthest left) contained a protein standard. The largest protein in thestandard (the band of the top of the gel) was 209,000 kDa, and the smallest was 7,100 kDa.

Lanes 2 and 3 show the darkest set of bands (proteins) around 46,000 kDa to 100,000 kDa.

Lanes 2 and 3 also show quite prevalent proteins at about 77,000 kDa, 82,000 kDa and100,000 kDa. Lane 5 shows almost no protein, except for very faint proteins at about

46,000 kDa and 100,00 kDa. All of the lanes (except standard) show a protein at about

46,000 kDa and 100,000 kDa. Except for lane 5 and standards, all the samples have a

protein at about 82,000 kDa. The largest proteins in lanes 2, 3, 6, and 7 at about 170,000kDa are discreet.

Discussion:

Figure 1 shows the increase in production of NADPH when IDH catalyzes the reduction ofisocitrate to -ketoglutarate over time, as the NADPH is absorbed by the light at 340nm.

The 35% S, flow-thru, and wash samples showed the biggest increases in absorbance, and

therefore had more IDH than the other samples. The almost flat slopes of the other

samples indicate that there was not much IDH in the samples.


5/5

To acquire the activity values in Table 1 for each sample, the data from time points 180 to

240 (Fig. 1) were used. This time frame was chosen because the slope was increasinglinearly. Also, the time frame contained absorbance values that were above the minimum

of the optical range of the spectrophotometer (0.12.0 for the spectrophotometer used,

absorbance values in this range are usable).

The specific activity was highest in the 60 mM KCl sample, yet it had the smallest non-

zero value for total protein. Since specific activity is a measurement of enzyme activityoccurring among a specific quantity of protein, the results would conclude that this sample

contained the most pure form of IDH.

Much protein was eluted in the flow-thru and wash steps. A decent amount of salt stillcould have remained in the initial solution after being dialyzed. If the solution were

dialyzed for a longer period, less salt would be in solution to inhibit the attachment of the

proteins to the resin.

In Figure 2, the smears of proteins are caused by too many proteins being in the sample.The biggest proteins were in lanes 2, 3, 6, and 7. The sample for lane 7, the 80 mM KCl

solution, however presented a zero value for the protein concentration.

MJohnson MMBB380 Report 4-5

Documents

Transcript of MJohnson MMBB380 Report 4-5