Isolation of Rubisco from Spinach Leaves

Isolation of Rubisco from Spinach Leaves

Introduction

The purpose of this lab is to isolate Rubisco from spinach leaves. Rubisco is being isolated because it is so essential to life. no specifics about your expt Rubisco, otherwise known as ribulose bisphosphate carboxylase/oxygenase, is essential because it takes the oxidized carbonCO2 from the earth and fixes it into an more organic form, rich with carbon-carbon double bonds and many hydrogen atoms which is???. This carbon fixation takes place within plant cells. Inside of the plant cells, Rubisco forms the bridge between the life and the lifeless, therefore creating organic carbon from the inorganic carbon dioxide in the airyou basically said this idea already. Though very abundant, Rubisco is remarkably inefficient, again, in theis single sentence you basically say the same thing twice therefore, plant cells compensate for this inefficiency by building more of this enzyme. There are many methods to isolateing proteins, but including again, no specifics….the methods that are used in this particular isolation include the Ammonium Sulfate Precipitation, the Ion Exchange Column Chromatography, and the SDS PAGE.

The Ammonium Sulfate Precipitation is a method of protein purification that works by changing the solubility of the protein. Ammonium sulfate is used because it has a high solubility, therefore solutions with high salt concentrations are allowed to do?. This ammonium salt inhibits the protein's interaction with the water why? How?, thus allowing the proteins to be drawn out of solution. The solubility of proteins depends on the ionic strength of the solution, as well as the salt concentration how does this relate to the previous info?. At low salt concentrations, the solubility of the protein increases with increasing salt concentration this doesn'tmake sense…. At high salt concentrations, the solubility of the protein begins to decrease, and at even higher salt concentrations, the protein will be completely precipitated from the solution.

The Ion Exchange Column otherwise known as Ion Exchange Chromatography no, these terms are not interchangeable…one is a piece of equipment and the other is a process is a process that allows the separation of ions and polar molecules based on their charge to occur??. This method involves two primary if there are primary, then what are the secondary??? steps, which are the binding of a protein to a charged resin, and the second step involves the elution or the displacement of the protein from the charges of the resin. The pH of the buffer is very essential to? because it is used to equilibrate and no, the pH does not load anything….the researcher does load the proteins onto the column, and it is also important for the elution to occur why? How?. This particular processno specifics of our expt…. requires an anion exchanger, thus DEAE-cellulose is used. DEAE-cellulose has a cellulose bead as the support material which has been derivatized with a positively charged amino group displaying? a positive charge into the solution?. Thus, what occurs is that, as the solution is being poured into the column, all the negatively charged proteins are going to be attracted to the resin. As the salt concentration is poured a concentration cannot be poured, a solution can be poured, the more soluble protein will be eluted, and as the salt concentration increases, the less soluble proteins will eventually come out of the solution. This process is not about solubility , its about charge……This process requires different fractions to be taken???, in order to determine which of them has the most protein why?.

Lastly the SDS PAGE is the final method used to isolate Rubisco from spinach leaves. The purpose of the SDS page is to again, no specifics…separate proteins according to their size. SDS, otherwise known as sodium dodecyl sulfate is a detergent that can dissolve hydrophobic molecules, but also has a negative charged attached to it. Therefore, if a cell is incubated with SDS, the membranes will be dissolved, all the proteins will be soluablized by the detergent, and the protein will be covered with many negative charges don't mention membrane dissolution since it has nothing to do with this expt.. Thus, what occurs is that the negatively charged proteins will be attracted to the positive pole when placed in an electric field. If the proteins are denatured treated with SDS and placed into an electric field, they will all migrate towards the positive pole at the same rate, with no separation by size. PAGE (polyacrylamide gel electrophoresis) therefore how? provides an environment that will allow different sized proteins to move at different rates. The acrylamide forms a gel, and electricity is used to pull the proteins through the gelthis sentence is a repeat of ideas and is vague. Thus, what occurs is that bands develop? They don't develop, they move? along this gel, indicating the presence of proteins that are separated based upon their size.

Materials and Methods

Ammonium Sulfate Precipitation: Approximately 300g of spinach leaves were deribbed use past tense. The leaves were homogenized in 200ml Buffer I. It was filtrated with miracloth, and about 40ml of ammonium sulfate was added to the solution slowly and placed on the stir plate, to obtain a 50%I thought you did 37% first? saturation. The solution was then centrifuged at 9000*g for 15 minutes. The pellet was resuspended in 4ml of distilled H2O, and placed in a dialysis bag, to allow for a semi-permeable membrane to get the salt contents out, and the protein to remain. Approximately 2.06g of ammonium sulfate talk about concentrations, not amounts…only concentrations are transferrable….was slowly added to the supernatant and was centrifuged at 9000*g for 15 minutes. The pellet was once again resuspended in 4ml of distilled H2O and placed in a dialysis bag. And then what?

Ion Column Exchange Chromatography: The columns were equilibrated with Buffer A ( 10mM Tris pH 8.0, 3mM EDTA). P1 was diluted a 100 fold, and 3ml of diluted P1 and undiluted P2 was loaded onto separate columns. The column was then washed with 10ml Buffer A. About 10ml of low salt buffer (Buffer A + 50mM Nacl) was then added to the columns, and 5 fractions were collected in cuvettes to measure their OD values at 280nm. Approximately 10ml of medium salt buffer (Buffer A +200mM Nacl) was added to the columns, and 5 fractions were collected in cuvettes to determine their OD values at 280nm. 10ml of high salt buffer (Buffer A + 500mM Nacl) was added to the columns and 5 fractions were collected in cuvettes to determine their OD values. For each, the cuvette with the highest OD level was saved and placed in ice for later use.

SDS PAGE: Approximately 60µl from each of the 9 samples ( homogenate, P1, P1 low, P1 med,I would prefer to see a more descriptive title like P37 and S37 and P P1 high, P2 low, P2 50, etc….med, P2 high) were transferred to?, and 30µl of 3X sample buffer what's in this? was added. Protein standards were obtained and all the samples were heated for 4 minutes at 95°C. The gel chamber was prepared made to a concentration of with 10% acrylamide in?, and the chamber was filled with running buffer ( Tris, glycine, and SDS give concentrations). Approximately 25µl of each sample was pippetted into each lane of the gel, and 10µl of the standard was loaded into a lane. The gel was ran at 180 volts for 50 minutes, and stained with staining solution what's in this? Destain? Dried? for about 30 minutes.

Results

The OD values for both pellet 1 and pellet 2 can be seen below in tables 1 and 2. The highlighted portions are the cuvettes that were saved to carry out the SDS PAGE. These values were chosen because they had the highest OD value, stating that protein must be present in one of the following. Give actual data (numbers) and then reference them with (Table 1) at the end of the sentence ….

Table 1. ODwavelength? levels??? Not sure what OD levels are… of Pellet 1samples taken after carrying out the ion exchange column chromatography

Pellet 1

OD Level

L1

-0.210

L2

-0.256

L3

-0.2262

L4

-0.209

L5

-0.188

M1

-0.206

M2

-0.222

M3

-0.030

M4

-0.020

M5

0.014

H1

0.044

H2

0.139

H3

0.119

H4

0.181

H5

0.176

Caption???

Table 2. OD levels of Pellet 2 after carrying out the ion exchange column chromatography

Pellet 2

OD Level

L1

0.197

L2

-0.182

L3

-0.011

L4

0..451

L5

0.007

M1

-0.024

M2

0.115

M3

0.064

M4

0.405

M5

1.118

H1

0.956

H2

0.502

H3

0.301

H4

0.102

H5

0.756

Discussion

The procedures were carried out with expectations We expected that the protein Rubisco be present in the standard (why?), pellet 1, pellet 1 med and high, as well as in the homogenate why?. After carrying out theSince we saw _____ in the SDS PAGE, it was concluded that Rubisco was present in the standard NO, Rubisco is NOT in the std…., pellet 1 high, pellet 2 med, pellet 2 high, and the homogenate. Pellet 2 was not included in the gels due to the fact that it was misplaced by the instructors. Due to this mishap, it was not possible to make a comparison between Pellet 1(37%saturation) and Pellet 2(50% saturation). Therefore, nothing can be concluded about the solubility of Pellet 2. Well, proteins in pellet 2 would be expected to be soluble…you just don't know if rubisco is there… Thus, it is not sure as to whether Rubisco is actually present, due to the absence of Pellet 2.

You didn't talk about the bands you say in terms of size, charge or solubility…. use these as the basis of your discussion….

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