How SDS PAGE Works: Step-by-Step Protocol and Student Tips
Proteins are essential biomolecules that drive a multitude of processes within living organisms. If you’ve ever wondered how scientists accurately separate and analyse these molecular machines, sds page (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis) is the key. This page breaks down sds-page principle, sds-page protocol, and sds-page application in a way that’s easy to follow—whether you’re a budding high school student or a college-level biology enthusiast.
What is the SDS-PAGE Full Form?
The sds-page full form stands for Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis. It is a specialised type of gel electrophoresis where proteins are separated predominantly based on their molecular weight. This technique eliminates the effect of the protein’s shape and intrinsic charge, making the separation process highly accurate for molecular weight estimation.
SDS-PAGE Principle Explained
The sds-page principle relies on two main components:
Sodium Dodecyl Sulphate (SDS): A strong anionic detergent that denatures proteins by breaking non-covalent bonds and coating them with a negative charge. As a result, proteins become linear polypeptide chains carrying a uniform negative charge.
Polyacrylamide Gel: A mesh-like matrix that acts like a sieve, allowing smaller proteins to move faster and larger proteins to move slower when an electric field is applied.
As the electric current passes through the gel, proteins coated in SDS migrate towards the positive electrode. Because their intrinsic charges and shapes have been standardised by SDS, their mobility depends mainly on their size. This sds page separation is precise and reproducible, making it the go-to method for analysing protein mixtures.
Also, read Principles of Biotechnology
Materials and Setup for SDS-PAGE
To perform sds-page protocol effectively, you need:
Power Supply: Converts AC current to a stable DC current.
Precast or Hand-Cast Gels: Polyacrylamide gels can be prepared in the lab or bought ready-made.
Electrophoresis Chamber/Tank: Holds the gel cassette and allows the buffer to surround it.
Protein Samples: Mixed with SDS-PAGE sample buffer (containing SDS and a reducing agent like dithiothreitol or 2-mercaptoethanol) and boiled to denature the proteins.
Running Buffer: Typically Tris-glycine-SDS buffer used to maintain pH and conductivity.
Staining and Destaining Solutions: Commonly Coomassie Brilliant Blue for staining protein bands, followed by a destaining solution to reveal clear bands.
Protein Ladder/Marker: A reference mixture of proteins with known molecular weights to estimate the size of your proteins of interest.
Step-by-Step SDS-PAGE Protocol
Understanding the sds-page protocol is crucial for reproducible results:
Gel Preparation
Prepare the separating gel solution by mixing acrylamide, buffer, and SDS. Finally, add TEMED (tetramethylethylenediamine) and ammonium persulphate (APS) to initiate polymerisation.
Pour the separating gel into the casting chamber.
Add a thin layer of butanol or isopropanol on top to level the gel and remove air bubbles. Once set, rinse off the top layer.
Prepare and pour the stacking gel above the separating gel. Insert the comb to form wells.
Sample Preparation
Add 2-mercaptoethanol or dithiothreitol to your sample buffer to break disulphide bonds.
Mix your protein sample with this buffer.
Boil for about 5 minutes to ensure complete denaturation.
Electrophoresis
Place the polymerised gel (the “gel cassette”) in the electrophoresis chamber.
Fill the chamber with 1x running buffer, ensuring the wells are fully submerged.
Carefully load your protein samples and the molecular weight markers into the wells using a pipette.
Close the lid and connect the chamber to the power supply. Set the current to around 30 mA for a typical mini-gel.
Run for approximately 1 hour or until the tracking dye reaches the bottom of the gel.
Staining and Destaining
After electrophoresis, remove the gel from the cassette.
Immerse it in Coomassie Brilliant Blue staining solution for 30 minutes to 1 hour.
Destain with an appropriate solution (often a mixture of methanol, acetic acid, and water) to visualise your protein bands clearly.
Analysis
Compare the mobility of your protein bands to the reference protein ladder.
Document the gel by taking a photograph or scanning.
Role of SDS and Reducing Agents
SDS coats polypeptides with a negative charge proportional to their mass, ensuring that proteins migrate primarily based on size rather than shape or intrinsic charge.
Reducing Agents (like DTT or 2-mercaptoethanol) break disulphide bonds, aiding in the complete denaturation of proteins. This ensures proteins do not refold or maintain subunit interactions during the run.
Also, read Applications of Biotechnology
Going Beyond the Basics
While sds-page principle focuses on size-based separation, here are some additional tips and advanced methods to make your results stand out:
Stacking vs Separating Gel: The stacking gel has a lower acrylamide concentration and an acidic pH, which compacts the proteins into tight bands before they enter the separating gel. This improves resolution.
Silver Staining: An alternative to Coomassie that offers higher sensitivity, ideal for detecting very low protein concentrations.
Fluorescent Labelling: Using fluorescent dyes can help in quantification and multiplexing several samples on a single gel.
2D Gel Electrophoresis: Combine isoelectric focusing with SDS-PAGE to separate proteins first by charge (pI) and then by molecular weight, giving an even more detailed profile.
SDS-PAGE Application
sds-page application is extensive in molecular biology, biotechnology, and medical diagnostics. Some common uses include:
Molecular Weight Estimation: Precisely determine protein size by comparing to known markers.
Protein Purity Check: Evaluate whether your sample contains contaminants.
Polypeptide Composition: Study subunit composition of complex proteins.
Peptide Mapping: Fragment proteins and separate them for structural analysis.
Post-Translational Modifications: Detect shifts in apparent molecular weight due to phosphorylation, glycosylation, etc.
Medical Diagnostics: Used in western blotting for HIV tests or other disease markers.
Protein Ubiquitination Studies: Identify ubiquitinated proteins by observing changes in band patterns.
Linking SDS-PAGE to Western Blotting
One of the most prominent techniques that follows sds page is western blotting, where proteins separated by SDS-PAGE are transferred to a membrane and probed with specific antibodies. For more details on how proteins are detected after sds-page protocol, visit our dedicated Western Blotting page on Vedantu (link placeholder).
Interactive Quiz: Test Your SDS-PAGE Knowledge
Which two main factors determine protein separation in sds page?
A. Size and shape
B. Shape and charge
C. Size and charge
D. Charge alone
What is the primary purpose of adding SDS in the sds-page protocol?
A. To colour the proteins
B. To standardise protein shape and charge
C. To enhance polymerisation
D. To cool the gel
In sds-page principle, why are reducing agents added?
A. To increase gel temperature
B. To break disulphide bonds
C. To stabilise proteins
D. To form SDS micelles
How do we typically visualise protein bands after sds-page application?
A. UV light alone
B. Boiling the gel
C. Staining with Coomassie or Silver stain
D. Adding agarose
Which method often follows SDS-PAGE for specific protein detection?
A. Northern blotting
B. Western blotting
C. Southern blotting
D. Eastern blotting
Check Your Answers
C
B
B
C
B
FAQs on SDS PAGE Explained: Principles, Steps & Uses in Protein Analysis
1. What does SDS-PAGE stand for?
SDS-PAGE stands for Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis. It is a powerful and widely used technique in molecular biology and biochemistry to separate proteins primarily based on their molecular weight.
2. What is the main principle of SDS-PAGE?
The main principle of SDS-PAGE is to separate proteins based almost exclusively on their molecular weight. The detergent SDS denatures proteins and provides a uniform negative charge, which masks the protein's intrinsic charge. When an electric field is applied, these negatively charged proteins migrate through the polyacrylamide gel towards the positive electrode. The gel acts as a molecular sieve, allowing smaller proteins to move faster and further than larger ones.
3. What is the specific role of the SDS detergent in this technique?
Sodium Dodecyl Sulphate (SDS) performs two crucial functions:
Denaturation: It disrupts the secondary, tertiary, and quaternary structures of proteins, unfolding them into linear polypeptide chains.
Uniform Charge: It binds to the protein backbone at a relatively constant ratio, imparting a strong, uniform negative charge that is proportional to the protein's mass. This ensures that separation is based on size, not the protein's native charge.
4. Why are both a stacking gel and a separating gel used in SDS-PAGE?
Using two different gels is a key example of how SDS-PAGE improves the resolution of protein bands.
The stacking gel has larger pores and a lower pH. Its purpose is to concentrate all the protein samples from the wide well into a single, sharp, narrow band before they enter the main separating gel.
The separating gel has smaller pores and a higher pH. This is where the actual separation of proteins occurs based on their size, resulting in distinct, well-resolved bands.
5. What would happen if a reducing agent was omitted from the sample buffer?
If a reducing agent like β-mercaptoethanol or DTT were omitted, the disulfide bonds within or between polypeptide chains would not be broken. This means the protein's tertiary or quaternary structure, which is held together by these bonds, would remain partially intact. As a result, the protein would not be fully linearized, its migration through the gel would be abnormal, and its apparent molecular weight would be incorrect.
6. How does SDS-PAGE differ from Native PAGE?
The key difference lies in the conditions under which proteins are separated, which highlights the importance of choosing the right technique for an experiment.
In SDS-PAGE, proteins are denatured. They are separated solely based on their molecular mass.
In Native PAGE, proteins remain in their folded, native conformation. Separation occurs based on a combination of factors, including the protein's intrinsic size, shape, and charge.
7. What is the role of TEMED and APS in preparing the gel?
TEMED (Tetramethylethylenediamine) and APS (Ammonium Persulfate) are essential reagents for creating the polyacrylamide gel. APS acts as the initiator, generating free radicals that start the reaction. TEMED acts as a catalyst, accelerating the rate of free radical formation and driving the polymerization of acrylamide and bis-acrylamide monomers into a cross-linked gel matrix.
8. What is the function of a protein ladder in SDS-PAGE?
A protein ladder, also known as a molecular weight marker, is a mixture of several well-characterised proteins with known molecular weights. It is loaded into one of the wells and run alongside the unknown samples. By comparing the migration distance of the unknown protein bands to the bands of the ladder, one can estimate the molecular weight of the sample proteins.
9. If a protein appears as a smear instead of a sharp band, what could be the possible reasons?
A smear instead of a crisp band is a common issue and can provide clues about the sample. Possible reasons include:
Protein Degradation: The sample may have been degraded by proteases, creating many smaller fragments.
Overloading: Too much protein was loaded into the well, causing diffusion and streaking.
Incomplete Denaturation: The protein was not fully unfolded, leading to inconsistent migration patterns.
High Salt Concentration: Excessive salt in the sample buffer can interfere with the electric field and cause poor resolution.
10. How are the results of an SDS-PAGE gel visualized and analysed?
After electrophoresis, the gel must be stained to visualize the separated protein bands, as they are otherwise invisible. Common visualization methods include:
Coomassie Brilliant Blue: A widely used, simple, and moderately sensitive stain.
Silver Staining: A more complex but highly sensitive method for detecting very low amounts of protein.
Analysis involves estimating protein size by comparing bands to the protein ladder and assessing the purity or expression levels of the protein.
11. Can SDS-PAGE determine the exact mass of a protein? Why or why not?
No, SDS-PAGE provides an estimation of a protein's molecular weight, not its exact mass. The estimation is based on its migration relative to the known standards in the protein ladder. Factors like post-translational modifications (e.g., glycosylation) or unusual amino acid compositions can affect how a protein binds to SDS and migrates, leading to a discrepancy between its apparent weight on the gel and its true molecular mass.
12. What are some key applications of SDS-PAGE in research and diagnostics?
SDS-PAGE is a fundamental technique with broad real-world applications, including:
Assessing Sample Purity: Checking for contaminants in a purified protein sample.
Monitoring Protein Expression: Comparing protein levels between different biological samples.
Identifying Proteins: It is the crucial first step in Western Blotting, a technique used to detect specific proteins, such as identifying viral proteins (e.g., HIV) in medical diagnostics.
