Isolating Rubisco Protein Using Ion Exchange And Electrophoresis Biology Essay

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Rubisco is a negatively charged protein that weighs 55,000 kDa and is also very soluble. When we add ammonium sulfate to reach a saturation of 50%, Rubisco can be isolated using ion exchange chromatography and protein electrophoresis. This would go at the end of the intro…its OK here.

Materials and Methods

Isolation of Protein by Ammonium Sulfate Precipitation

Approximately 300g of fresh spinach leaves were de-ribbed and dried, then homogenized for 1 minute in 200mL of buffer. From the homogenized solution, approximately 50ml was placed into a beaker, which was stirred on a stir plate while 10.90g of solid ammonium sulfate was slowly added to reach 37% saturation. The solution was stirred for an additional 10 minutes and then centrifuged at 9,000-xg for 15 minutes. The pellet (Pellet I) was resuspended in 4mL of water and transferred into a dialysis bag for dialysis against distilled water. The supernatant was poured into a beaker and stirred on a stir plate while about 3g of ammonium sulfate was slowly added to reach 50% saturation. After about 15 minutes of stirring, the supernatant was centrifuged for 15 minutes at 7,000-xg. Following centrifugation, the pellet (Pellet II) was resuspended in 4mL of water and transferred into a dialysis bag for dialysis.

Ion Exchange Column Chromatography

The column was equilibrated by running 30mL of Buffer A (10mM Tris pH 8.0, 3mM EDTA) through. Afterwards, a centrifuge was used to pellet down any solid precipitate out of the dialyzed samples. 1mL of each sample (Pellet I and II) was transferred into labeled Eppendorf tubes and frozen for later use in the SDS-PAGE. Pellet I was diluted a 100-fold and about 4mL of diluted Pellet I and undiluted Pellet II were loaded into separate columns and the samples were allowed to flow through. The flow-through was discarded.

10mL of the low salt buffer (Buffer A + 50mM NaCl) was loaded onto the column and fractions of approximately 2mL were collected in separate cuvettes and labeled in order. After blanking the spectrophotometer at 280nm with low salt buffer, the OD readings of each fraction was measured. This process was repeated using the medium salt buffer (Buffer A + 200mM NaCl) and high salt buffer (Buffer A + 500mM NaCl). The spectrophotometer was blanked with each buffer before the readings of its corresponding fractions were obtained. The fractions with the highest OD reading at 280nm was collected in an Eppendorf tube, labeled, and placed on ice. The column was then washed with 10mL resin cleaning buffer which was discarded in a wash beaker.

Protein Electrophoresis

30uL of 3X Sample Buffer (bromophenol blue, glycerol, dithiothreitol, and SDS) was added to 60ul of each of the 9 samples (standard, homogenate, Pellet I, Pellet I low, medium and high salt buffers, Pellet II low, medium and high salt buffers). The 9 tubes were heated in a water bath for 4 minutes. Into a prepared buffer chamber, approximately 20uL of each sample were loaded into separate wells using thin-barrel pipette tips. The gel was run at 180 Volts for approximately 50 minutes. The gel was removed from the glass plate sandwich and stained in Staining/Fixing Solution for 30 minutes. The gel was destained overnight in Destaining Solution and then dried ona vacuum gel dryer for one hour.

Results

Table 1: ODwavelength Readings of Protein Fractions for Pellet I

OD Reading (280nm)

Tube

Low Salt

Medium Salt

High Salt

1

0.202

-0.077

0.020

2

0.162

0.022

-0.083

3

-0.015

-0.077

-0.059

4

0.246

0.003

-0.068

5

0.002

0.020

0.052

*OD readings (280nm) for the fractions were measured using a spectrophotometer after they eluted from the ion exchange column.

Table 2: OD Readings of Protein Fractions for Pellet II

OD Reading (280nm)

Tube

Low Salt

Medium Salt

High Salt

1

HI

0.121

-0.069

2

HI

0.687

-0.059

3

1.442

-0.017

-0.095

4

0.963

0.025

0.200

5

0.229

-0.049

0.320

*OD readings (280nm) for the fractions were measured using a spectrophotometer after they eluted from the ion exchange column.

Table 3: Known Protein Standards, Relative Molecular Weights and Distance Traveled on Gel

Proteins

Distance Traveled (mm)

Molecular Weight (Daltons)

Phosphorylase b

80

97,000

Albumin

130

66,000

Ovalbumin

200

45,000

Carbonic anhydrase

260

30,000

Trypsin Indicator

--

20,100

Lactalbumin

--

14,400

*The molecular weights of the proteins are known and the distance traveled was gotten by measuring with a ruler from the top of the well to the band of each band.

Figure 1: Standard Curve of Known Protein Weights Vs Distance Traveled on Gel

Weight (kDa)

*Solutions of proteins with known weights were loaded and run in SDS-PAGE and the distance was measured from the top of the well to the bottom of the band.

Table 4: title

Sample #

Sample

No. of bands

Migration distance (cm)

Molecular Weight (kDa)

1

Pellet I Low salt

-

-

-

2

PI Medium salt

-

-

-

3

PI High salt

-

-

-

4

PI

1

2.7

28

5

Pellet II Low salt

1

2.8

26

6

PII Medium salt

-

-

-

7

PII High salt

-

-

-

8

Standard

1

2

3

4

0.8

1.3

2

2.6

97

66

45

30

9

Homogenate

1

2

3

1.8

2.3

2.6

50.3

36

30

*The number of bands were tallied, then the molecular weight was collected as well as the migration distance. The migration was found by measuring from the top of the well to the bottom of the band.

Four bands showed in the lane where the standard was loaded instead six, because there were six proteins present in the sample this does not make sense to me. Three bands give sizes appeared in the homogenate and one band in Pellet I and Pellet II low salt.

Present all of your data….

Discussion

Rubisco weighs 55,000 kDa according to literature and gel bands that correlate with that size did not appear in either of the samples. It was predicted Rubisco is very negatively charged and very soluble, therefore it should elute the column at a high salt concentration because a large concentration of salt should be needed to disrupt the bonds created between the negatively charged ion of Rubisco and the positively charged resin, but the high salt buffer fractions had some of the lowest OD readings which is conflicting with our prediction. Instead, the OD readings were highest with the low salt buffer concentration. This could mean that Rubisco is not as negatively charged and soluble as we predicted. I cannot clearly conclude if Rubisco was isolated or not because the gel ripped and a big chunk of it was missing due to mishandling. Also, Rubisco might have been lost due to contamination because they were no bands that correlated with its molecular weight. Good!

The quality of the experiment could be improved by some additional methods to identify Rubisco since it fixes carbon dioxide. Therefore they could be a test to show that the protein that was isolated can actually fix carbon dioxide.

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