Halit Koçak PROTEIN ISOLATION AND SDS-PAGE 26326

Introduction:

Along the lines of improving the perception of protein function and interaction, many techniques
have been designed to separate and analyze proteins with higher resolutions. Significantly, there
is the Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), one of the key
methods for the separation of proteins from complex mixtures on the basis of molecular weight.
This technique uses an SDS anionic detergent that binds to the polypeptide chain of proteins by
denaturing them and giving a negative charge that is proportional to the length of the polypeptide
chain. This process of denaturation unfolds the protein completely; hence, this will help to
reduce the variability in shape and allow the deciding factor for size only in the process of
electrophoresis.

This may well be continuous or discontinuous, which again would make the system more
effective for SDS-Poefficient-gel electrophoresis. Proteins are first concentrated in a thin layer at
the top of a stacking gel before getting resolved by size in the separating gel.

That would make this approach separate proteins according to their molecular weight with high
resolution. It also allows detail analysis on the purity of the protein and the molecular weight;
therefore, it is very valuable for research works in the field of biochemistry and molecular
biology. Traditional SDS-PAGE has disadvantages, such as the failure to maintain the
conformation of the native state protein throughout SDS-PAGE, a situation that is, however, very
imperative for functional studies. Denaturing conditions affect non-covalent bonds and,
moreover, protein complexes, which are therefore frequently needed to unveil information
relating to the activity and interactions of proteins. This limitation is targeted to be overcome,
and some modifications are sought for ensuring better retention of protein functionality while at
the same time gaining high resolution in separation, for example, by reduction in the
concentration of SDS in the running buffer.

Materials:

1. Bacterial cultures

2. Sonication device

3. Refrigerated centrifuge
Halit Koçak PROTEIN ISOLATION AND SDS-PAGE 26326

4. Lysis buffer (specific composition not listed)

5. SDS-PAGE equipment:

• Gel casting apparatus

• Power supply

6. Acrylamide/bis-acrylamide solution

7. Tris buffer (for preparing stacking and resolving gels)

8. SDS solution (for running buffer and gel preparation)

9. Ammonium persulfate (APS, for initiating polymerization)

10. Tetramethylethylenediamine (TEMED, for accelerating polymerization)

11. Protein ladder (standard molecular weight markers)

12. Coomassie Brilliant Blue R-250 (for staining proteins)

13. Destaining solution (methanol-acetic acid-water mixture)

14. Gel documentation system (for imaging and analyzing gels)

Methods:

1. Assembling the SDS-PAGE System

- The gel apparatus was set up following a thorough cleaning with ethanol and water.

- Glass plates and combs were also cleaned with ethanol to ensure removal of any
contaminants.

2. Sonication

- For the lysis of bacterial samples, sonication was carried out for 3 cycles of 20 seconds each,
interspersed with 30-second rest intervals.

- The samples were then centrifuged at 13,000 x g for 5 minutes.

Halit Koçak PROTEIN ISOLATION AND SDS-PAGE 26326

- The resulting supernatant was carefully retrieved, avoiding the cell debris pellet.

Figure 1 is indicating the sonication device

Figure 1: Sonication Device

3. Preparation of the Resolving and Stacking Gel

- The resolving gel was prepared with a final acrylamide concentration of 12%, using 1.5M
Tris-HCl (pH 8.8), 10% SDS, water, 10% APS, and TEMED. The gel was allowed to polymerize
before adding the stacking gel.

- The stacking gel had a final acrylamide concentration of 4% and was prepared using 0.5M
Tris-HCl (pH 6.8), 10% SDS, water, 10% APS, and TEMED.

Figure 1 is showing the step that is mixing the gel.

4. Preparation of Samples

- Each protein sample was mixed with 4x SDS sample buffer.

Halit Koçak PROTEIN ISOLATION AND SDS-PAGE 26326

- The mixture was then heated to 100°C for 5 minutes to ensure complete denaturation of the
proteins.

5. Sample Loading

- After carefully removing the comb, the gel wells were rinsed with running buffer.

- Samples were loaded into the wells, alongside the molecular weight standards.

Figure 2: Sample loading process with accurate hand positioning

6. Running the Gel

- Electrophoresis was conducted at 80V for the stacking gel, then the voltage was increased to
120V for the resolving gel.

- The run was continued until the dye front reached the end of the gel, a process that took
approximately 3.5 hours.

7. Staining and Documentation

- Following electrophoresis, the gel was stained with Coomassie Brilliant Blue to visualize the
protein bands.

- After sufficient staining, the gel was destained to clear the background.
Halit Koçak PROTEIN ISOLATION AND SDS-PAGE 26326

- The resulting gel was documented for analysis of protein separation.

Results:

SDS-PAGE analysis was carried out to separate the proteins isolated from the bacterial cultures.
The proteins are separated on the gel according to their molecular mass and subsequently
visualized with Coomassie Brilliant Blue staining as shown in figure 3.

Figure 3: Result image for SDS-PAGE

1. Protein Ladder (Lane 1): An MW standard was loaded in this lane. The bands
appeared with the expected size of the sample proteins against the protein ladder.

2. Blank Rosetta (Lane 2): No significant protein bands were observed in the blank
control.

3. Lysed AFP (Lane 3): Lane 3 of lysed AFP: In this lane, numerous bands have
been visualized, indicating that an array of proteins may be present. 4. Flowthrough of AFP
Halit Koçak PROTEIN ISOLATION AND SDS-PAGE 26326

(Lane 4): This is actually quite interesting because the proteins of all kinds are present in the lane
with the lysed sample, meaning that not all the proteins have been retained by the column.

5-10. AFP with imidazole gradient (Lanes 5-10): As the concentration of imidazole was
increased to 500, 750, and 1,000 mM, there was a clear progressive increase in purity of protein
eluted through the affinity column with the intention to elute the protein of interest.

With a much clearer protein band matching the estimated size of the target protein, especially at
lanes 9 and 10 (150 mM and 200 mM imidazole, respectively), one can see that at higher
concentrations of imidazole, a distinct advantage is created. This band is marked by a red
rectangle in lane 9, indicating successful elution of the protein at this concentration. From the
above, the results mention that sonication effectively lyses a cell and shows various proteins.
Affinity purification enriches more the protein of interest in this method, especially at higher
imidazole concentrations. The intensity and sharpness in bands of lanes 9 and 10 both correlate
directly with the purity of the target protein. This becomes evident experimentally from controls
and gradients for further interpretation of the process of protein purification.