This experiment used the SDS-PAGE gel technique to separate proteins of interest. The presence of SDS serves to unfold proteins and allow the samples to carry charge.
Materials and Methods
1. Electrophoresis buffer from packet:
a. 500mL of Tris at 100mM
b .3mM SDS of pH 8 ± 0.5
c. 500 mL H2O
2. Gel, Tris HEPES SDS
a. 8-16% Precices Protein Lowell gel
3. SDS sample loading buffer (6x Reducing)
a. 375 mM, pH 6.8
b. SDS 6%
c. Glycerol 48%
d. Bromophenol 0.03%
e. MeSH 9%
4. 50/10 fixing solution 500 mL
a. 50% v/v methanol (250 mL)
b. 10% v/v acetic acid (50 mL)
5. Staining solution:
a. Coomassie Brilliant Blue R-250, 0.4 g in 200 mL of 50/10 de-staining solution
6. 10/10 de-staining solution, 200 mL
a.10% v/v methanol (20 mL)
b. 10% v/v acetic acid (20 mL)
c. 180 mL H2O
7. Unknown protein: pre-prepared
8. Protein markers: pre-prepared
A pre-cast protein gel was placed into a electrophoresis tank system apparatus, the inner and outer tanks were filled with SDS-page running buffer, and the gel was pre-electrophoresed for approximately 10 minutes.
10 μL of unknown protein(s) and 10 μL SDS sample buffer were transferred into an eppendorf tube, while 5 μL of protein marker and 5 μL of SDS sample buffer were transferred into a separate eppendorf tube. Both tubes were mixed and placed in a boiling water bath for approximately 3 minutes. The protein gel sample wells were rinsed several times with the running buffer using a transfer pipet to remove any contamination or air bubbles. Once prepared, the above protein samples were loaded into separate wells, the “gel rig leads” were connected to the system power supply and the gel was electrophoresed at a voltage of 100V for an approximate run time of 2 hours. After electrophoresis, the power supply was disconnected, the gel was removed from the apparatus, washed three times with water to remove excess running buffer, placed into a tray and stained for approximately 1 hour under constant agitation using Imperial protein stain. After 1 hour, the stain was removed; the gel was rinsed with water and placed into a water bath to de-stain for approximately 3 hours, under constant agitation, with a kimwipe in place to assist in stain particle removal. The gel was then photographed.
Results
The developed electrophoresis SDS-page gel in Figure 1 shows the unknown proteins, loaded into lanes 1,2,3,4 & 5. The known MW standard protein markers can be seen in lanes labeled ‘Ladder.’ Figure 2 shows an image of the known MW standard markers that were was used to generate the MW values for determinations of unknowns.
Ladder 1 2 3 4 5 Ladder
Figure 1: SDS Gel with ladder on both ends. Unknowns are labeled as lanes 1,2,3,4 & 5. Bands are visible for unknowns 1,2,3 & 5. Unknown 4 did not appear in the gel. Unknown 1 displays a band at around 35 and 25 kDa. Unknown 2 displays a band at 70 and 55 kDa, Unknown 3 displays a band at 35 kDa. Two bands are observed for unknown 5, one at 35 kDa and other at 98 kDa.
Figure 2: Ruler of distances in kDA for determination of standard distances on test gel
After using the PageRuler to determine the MW of the unknowns they were identified according to the list on the possible unknowns from Table 1 via matching unknown with a known that displayed the same positional residence.
Table 1. Known Molecular Masses, in kDa, of proteins.
Conclusions
Based on comparison to the standard knowns and then evaluated with regard to the list of known protein masses provided, unknowns were determined to be:
Unknown 1: Carbonic Anhydrase, 29 kDa
Unknown 2: Albumin, bovine, 63 kDa
Unknown 3: Albumin, egg, 45 kDa
Unknown 4: No Data
Unknown 5: Polymerase beta, 98 kDa
Unknown 4 was not able to be determined, as it did not appear in the final gel. This could occur for several reasons: The sample may have not been loaded, the provided unknown could have been contaminated, the unknown could have been mistakenly loaded outside of the well.
Questions
6.1)
• Unknown 1: Carbonic Anhydrase, 29 kDa
• Unknown 2: Albumin, bovine, 63 kDa
• Unknown 3: Albumin, egg, 45 kDa
• Unknown 4: No Data
• Unknown 5: Polymerase beta, 98 kDa
6.2)
The protein could be structurally denatured and then exposed to beta mercaptoethanol. The BME will reduce and cleave present disulfide bonds, resulting in the protein unfolding. If the protein was identically oligomeric, the subunits would separate into individual units. Smaller units would travel farther as they would have a reduced molecular weight. If a subunit was a dimer, the protein untis would now travel twice the distance as individual units than they would have as the dimer unit.
6.3)
Using BME in the same manner, new individual units would be formed from the cleaving of the disulfide bonds. After running a gel with the restructured proteins and compared to a gel ran with the same proteins, uncleaved.