Identifying Movement of DNA Proteins in Gel Electrophoresis Through Experimentation

Materials and Methods

Preparation of Spinach Homogenate

The experiment began by creating a homogenate from spinach leaves. This portion of the experiment was conducted by chopping 8g of spinach leaves as finely as possible. The leaves were then transferred to a cold mortar containing 12mL of buffer (.33M sorbitol, .05M HEPES, 5nM MgCl2, 2mM EDTA with a pH of 6.5). The leaves were then grounded into a fine paste. The paste was then filtered through cheesecloth with more buffer to remove large pieces of spinach

Differential Centrifugation

The researchers gathered 200μL of homogenate from the 1.5mL store and placed aside. The remaining homogenate was then centrifuged for 2 minutes at 200g. The supernatant was then transferred to a new tube. 200μL and supernatant was the collected, placed in a centrifuge tube labeled LSS (low speed supernatant) and placed on ice. The remaining supernatant was then centrifuged for 10 minutes at 1000g. The new supernatant was then transferred to a new centrifuge tube. 200μL was again removed and placed in a tube labeled HSS (high speed supernatant) and placed on ice. Any remaining supernatant was removed so that the pellet was completely dry. 250μL of homogenate was then used to resuspend the pellet via vortex. 200μL of the new suspension was transferred to a tube labeled HSP (high sped pellet) and place on ice.

Preparation of Fractions for SDS Polyacrylamide Gel Electrophoresis

The researchers gathered and labeled eight 1.5mL centrifuge tubes; CH-A, CH-B, LSS-A, LSS-B, HSS-A, HSS-A, HSP-A AND HSP-B. 50μL of each appropriate fraction was pipetted into each tube. To all of the tubes the researchers added 50μL of 2X Laemmli buffer. The tubes were all vortexed and then heated at 95°C for three minutes.

Determination of the Amount of Protein in Each Fraction

The researchers obtained five 13x100 test tubes and labeled them; blank, CH-1, LSS-1, HSS-1, HSP-1. 1mL of DI water was added to each tube along with 2μL of the appropriate fraction to tubes 2-5. The tubes were then vortexed and 1mL of coomassie dye was added to each tube. The tubes were all vortexed once more and tested on the spectrophotometer at 595nM.

Preparing Gel Apparatus

To set up the gel apparatus the researchers removed the gel from its packaging and removed the comb. The gel and wells were rinsed with DI water and the gel was placed into its slot in the apparatus. The gel box was then filled with 1X running buffer.

Preparing the Samples

The samples prepared with buffer were heated for 1-2minutes to solubilize the SDS. The samples were briefly centrifuged to gather all of the materials to the bottom. The appropriate amounts of each 1X Laemmli buffer, RUBISCO standard and protein were added to 12 centrifuge tubes according to tables 3a.3 and 3b.1.

Loading the Samples

The samples were loaded by placing the pipette tip deep into the well without touching the walls of the well. The power was then turned on to run the gel.

Removing and Staining the Gel

The power was disconnected from the box and the gel was carefully removed as to not tear it. The gel was then placed on a plate and stained with coomassie blue. The gel was allowed to sit so the stain could be set in. The gel was then examined under a UV light.

Table 3a.3 Columns C and D of this table show amount of mixture that was added to the centrifuge tubes to be used in the gel.

Table 3b.1 This Table shows which mixture was loaded into each of the 12 wells of the gel.

Results

Table 3b.3 This table shows the measured end bands for the 8 standard lanes of the gel electrophoresis.

Sample Migration Rf Molecular Mass

Standard 1 3/11 .27 110

Standard 2 2/11 .18 100

Standard 3 9/11 .82 90

Standard 4 9.1/11 .83 80

Standard 5 9.7/11 .88 70

Standard 6 9.8/11 .89 60

Standard 7 10/11 .90 50

Standard 8 10.1/11 .91 40

Figure 1 This figure is the standard curve calculated with the Rf values and the measured molecular weights.

Figure 2 This figure is a photo of the gel after the electrophoresis was fully run.

RUBISCO Subunit This table shows the calculated molecular weights from the standard curve for each band of the standard mixtures.

Lane Band Migration (cm) Rf Molecular Weight (Da)

CHa 1 1.5 .14 110785

2 8 .73 77238

CHa 1 1.5 .14 110785

2 8 .73 77238

HSSa 1 1.4 .13 111354

2 4.5 .41 95433

3 7.8 .71 78375

HSPa 1 1 .09 113628

2 4 .36 98276

3 6.5 .59 85198

CHb 1 1.2 .11 112491

2 3.8 .35 98845

LSSb 1 .9 .08 114197

HSSb 1 .8 .07 114765

HSPb 1 1.2 .11 1122491

2 3.5 .32 100550

3 4.5 .41 95433

4 8.5 .77 74963

Discussion

In the gel electrophoresis DNA proteins will always go towards the positive pole. Bromophenol blue has a slightly positive charge so therefore it will also move towards the positive pole. The molecular weights of the RUBISCO subunits range between 74Da to 114kDa. The banding patterns loaded in the Laemmli B are much closer together than those in A. The molecular weights of the subunits loaded in B range from 75kDa to 112kDa. It would appear that buffer B is the buffer without BME. The reason for this is because the lanes with the buffer B have more slow bands than those with buffer A. The slower bands are caused by larger subunits that the BME would have broken up if it were present. RUBISCO subunits do form disulfide bonds. This can be determined by looking at the lanes of the subunits with the buffer B. The slower bands in buffer B are larger subunits; these larger subunits that exist can only exist if disulfide bonds are holding the subunits together. The fraction LSS-B has the highest concentration of RUBISCO. This fraction can be found to have the highest amount of RUBISCO based on the high number of bands in that fraction’s lane. These bands could be compared to literature bands of RUBISCO that have been determined by accurate testing. If the bands in this gel match any bands of literature gels it will be clear that they contain RUBISCO. The fractions of CH-B and HSS-B both have bands that are not matched in the other fractions. The molecular weights of these bands are 98kDa and 114kDa. These bands are likely broken up chunks of DNA that is not RUBISCO but rather other small chunks of protein and DNA.