SDS-PAGE Analysis of Nuclear and Cytoplasmic Proteins

Principles and Mechanism of SDS-PAGE

Context and Purpose: This practical follows the separation of the nuclear fraction ( $N1$ ) and the cytoplasmic fraction ( $S1$ ) from chicken blood erythrocytes. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) is employed to separate and compare proteins from these two fractions.
Mechanism of SDS (Sodium Dodecyl Sulphate): * SDS is a charged detergent consisting of a negatively charged sulphate group attached to a long hydrophobic hydrocarbon chain. * The hydrophobic tail interacts strongly with the hydrophobic amino acid side chains of the protein, causing the protein to unfold into an extended chain structure (denaturation). * SDS coats the protein chain independently of its specific amino acid composition. * Binding Ratio: On average, one SDS molecule (carrying a negative charge) binds to every 2 amino acid residues. * Result: The native charge of the protein is overwhelmed (swamped). All proteins acquire a uniform net negative charge directly proportional to the length of the amino acid chain.
Separation by Electrophoresis: * Electric Field: When placed in an electric field, SDS-coated proteins migrate toward the positive electrode (the anode). * Sieving Effect: The polyacrylamide gel matrix acts as a sieve. Small pores retard the movement of larger proteins, while smaller proteins travel faster. * Mobility: Separation is based solely on size (molecular weight) rather than native charge or shape. * Banding: Each protein produces a band on the gel. The distance traveled is used to estimate molecular weight ( $MW$ ) by comparing migration to known molecular weight standards (markers) or to assess purity via band intensity.

Practical Aims and Laboratory Safety

Practical Aims: 1. To demonstrate the principles and practice of SDS-PAGE. 2. To analyze and compare proteins found in the cytoplasm and nucleus of chicken erythrocytes. 3. To determine the molecular weight of unknown proteins by comparing their migration with standards (completed post-staining).
Safety and Conduct: * Familiarize with COSHH (Control of Substances Hazardous to Health) and risk forms. * Maintain a clear and tidy workstation; write the lab book as the work progresses. * Waste Disposal: Glass and plastic should go in labeled bins; pipette tips go in bench containers. * Equipment must be returned to its origin, and work surfaces must be wiped down after use.
Hazard Warnings: * SDS loading buffer contains mercaptoethanol, which has a strong, obnoxious smell. * Methanol (used in staining) is toxic. * Gel plates are expensive, fragile (specifically the "ears" on the front plate), and become slippery when wet with detergent.

Technical Preparations and Pipetting Procedures

Buffer Stocks Reconstruction: * Nuclear Wash Buffer (1x NWB): Prepared from 10x stock. Final composition: $80\,mM\,NaCl$ , $10\,mM\,Tris-HCl\,pH\,7.5$ , $6\,mM\,MgCl_2$ . Final volume required = $5\,ml$ . * SDS Gel Running Buffer (1x): Prepared from 10x stock. Final volume required = $500\,ml$ .
Pipetting Best Practices: * Ensure tips are firm but not over-jammed. * Tip Depth: Never submerge the tip more than $2-3\,mm$ below the liquid surface to prevent excess sample sticking to the outside (which can cause up to $100\%$ volume error). * Plunger Use: Depress to the first stop to take up liquid. Depress slowly to the second/final stop to dispense into the bottom of the tube. * Mixing: Flick the tube to mix and give a brief ( $2-3\,sec$ ) spin in the centrifuge to collect liquid at the bottom.

Part 1: Preparation of Protein Samples

Nuclear Fraction (N1) Processing: 1. Thaw $N1$ at room temperature, then keep on ice. 2. Add $1\,ml$ of $1x\,NWB$ to the nuclear suspension. 3. Mix by pipetting up and down $2-3$ times. 4. Centrifuge for $12\,s$ at $10000g$ . (Ensure the rotor is balanced with a tube of equal volume opposite the sample). 5. Confirm a clear supernatant and a visible nuclear pellet; discard the supernatant. 6. Resuspend the pellet in $150\,\mu{l}$ of $1x\,NWB$ using a $100\,\mu{l}$ pipette.
DNA Digestion: Add $10\,units$ of $DNase1$ ( $5\,\mu{l}$ ). Incubate at $37^\circ C$ for $30-60\,minutes$ . This breaks down DNA to release proteins and reduce the viscosity of the solution for easier pipetting.
Loading Buffer Addition: * Add $100\,\mu{l}$ of 3x SDS loading buffer to the $N1$ suspension. * Composition of 3x SDS Loading Buffer: $9\%$ SDS, $15\%$ mercaptoethanol (reduces disulphide bonds), $15\%$ glycerol (increases density for loading), $0.004\%$ bromophenol blue (BPB tracking dye), and Tris-HCl buffer.
Cytoplasmic Fraction (S1) Processing: Transfer $20\,\mu{l}$ of $S1$ to a fresh tube and add $10\,\mu{l}$ of $3x\,SDS\,loading\,buffer$ . Mix well.
Heat Treatment: Spin samples to remove bubbles, then heat at $65^\circ C$ for $10\,minutes$ in a hot block to ensure complete reduction of disulphide bonds.

Part 2 & 3: Gel Assembly and Loading

Gel Assembly: * Use a $15\%$ SDS-acrylamide gel. * Remove the silicon rubber gasket seal from the sides/bottom of the plate (keep them as they are reusable). * Mark the bottom edge of the gel with a permanent marker. * Mount plates on the stand with the cut-out facing inward. Secure with the H-shaped clamping plate to form a water-tight seal. * Fill the bottom reservoir ( $2/3$ full) and top central reservoir with $1x\,running\,buffer$ . Ensure the buffer is $5-10\,mm$ above the plate cut-out. * Remove air bubbles under the gel using a syringe with a bent needle.
Loading Protocol: * Avoid the two narrow outer lanes to prevent "edge effect" distortion. * Load the EZ-Run molecular weight marker ( $5\,\mu{l}$ ) into a middle lane. * Wash the gel loading tip between different samples by pipetting up and down $3-4$ times in the bottom reservoir buffer. * Individual Sample Loads: 1. $3\,\mu{l}$ cytoplasmic proteins ( $S1$ ). 2. $6\,\mu{l}$ cytoplasmic proteins ( $S1$ ). 3. $3\,\mu{l}$ nuclear proteins ( $N1$ ). 4. $6\,\mu{l}$ nuclear proteins ( $N1$ ). 5. $9\,\mu{l}$ nuclear proteins ( $N1$ ).

Part 4: Electrophoresis and Staining

Power Supply Settings: * Terminal Connection: Cathode (black/negative) to the upper central reservoir; Anode (red/positive) to the bottom reservoir. * Settings: Constant voltage at $200\,V$ . * Troubleshooting: If current is $0\,mA$ , check lead connections and buffer levels. If current is markedly higher than $40\,mA$ , check for leaks or cracked plates.
Running the Gel: * Observe the Bromophenol Blue ( $BPB$ ) dye migrating toward the anode. If it moves upward, the electrodes are reversed. * Wait for the dye to reach the resolving gel; record band sharpening observations. * Stop the run when the $BPB$ dye is approximately $2\,mm$ from the marked end line.
Staining Procedure: * Stain Solution: $0.1\%$ Coomassie blue, $20\%$ methanol, $5\%$ acetic acid. * Gel Removal: Prise plates apart using a green gel clip. Cut the gel along the spacers to prevent tearing before lifting. * Staining Time: Overnight or at least $3\,hours$ . Gels are subsequently destained in acetic acid and methanol solutions.

Part 5: Analysis and Molecular Weight Determination

Calibration: 1. Identify the $6$ bands in the EZ marker lane. 2. Measure the distance from the interface of the stacking/running gel to the center of each marker band. 3. Plot a graph: Migration distance ( $x$ -axis) vs. $\log_{10}(MW)$ ( $y$ -axis). 4. Draw a best-fit trend line (calibration curve).
Estimation of Unknowns: * Identify the major band in cytoplasmic lanes and the $5$ major bands in nuclear lanes. * Measure their migration distances and use the calibration curve to find the corresponding $\log_{10}(MW)$ . * Convert the log value to the apparent molecular weight using the formula $MW = 10^{\text{intercept}}$ .
Data Recording: Document final voltage, current, and total run time. Note any deviations of proteins from the straight-line relationship on the graph.