Introduction
SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) and Western blotting are complementary techniques used to analyze proteins. While SDS-PAGE is a fundamental technique for separating proteins by molecular weight, western blotting allows for the transfer of these proteins onto a membrane, enabling specific identification. Together, these techniques provide a powerful approach for studying protein expression, structure, and function.
However, issues such as poor protein transfer, weak signals, or high background noise can compromise data quality, hindering the reliability and reproducibility of results. Using high-quality reagents, such as those supplied by Boston BioProducts, alongside an optimized protocol can minimize the risk of these common challenges.
Our SDS-PAGE and Western blot protocol offers detailed, step-by-step instructions, troubleshooting tips, and our best practices to help you obtain reliable, high-quality data.
Materials & Reagents
Here's everything you need to carry out our SDS-PAGE and western blot protocol. In addition to Boston BioProducts' high-quality buffers and reagents, you'll need the following materials and equipment:
- Pipettes and tips
- Microcentrifuge tubes
- Heat block (or water bath)
- Electrophoresis chamber and power supply
- Western blot transfer system
- Membrane (PVDF or nitrocellulose)
- Antibodies (primary and secondary)
- Imaging system
Boston BioProducts' Protocol
This SDS-PAGE and western blotting protocol will guide you through the key steps from sample preparation to detection, to help ensure clear and reproducible data and western blotting success!
Sample Preparation
Proper sample preparation is critical for obtaining reliable and reproducible Western blot results. This step ensures that proteins are extracted efficiently, remain intact, and are accurately quantified before SDS-PAGE. Below, we outline the key steps to prepare your samples for analysis.
Collect and label your samples carefully, and dilute them as necessary.
Remember to keep your samples on ice to prevent degradation!
Choose a lysis buffer based on your sample type and desired downstream applications (check out our recommendations). Add it to your samples to lyse them.
Remember to add protease inhibitors.
Choose the appropriate lysis buffer: NP-40 for whole cell lysate; RIPA membrane-bound, nuclear, or mitochondrial proteins; Tris-HCL for cytoplasmic proteins.
Centrifuge your samples to remove insoluble debris (we use 14,000 x g for 15 minutes for cell samples).
Then, transfer the supernatant to a clean microcentrifuge tube and discard the pellet.
Measure the protein concentration of your lysate using an appropriate assay, such as Bradford or BCA, to ensure equal loading across samples.
Use BCA assay if working with samples that contain <5% detergent for higher sensitivity.
Prepare your protein samples for SDS-PAGE by taking the recommended protein amount (10-50μg/lane) and diluting it in a sample buffer such as Laemmli. Heat the samples at 95°C for 5 minutes to fully denature the proteins.
SDS-PAGE
SDS-PAGE is a widely used technique to separate proteins based on molecular weight. The process involves denaturing proteins with SDS, which imparts a uniform negative charge, allowing proteins to migrate through a polyacrylamide gel under an electric field. The following steps guide you through gel preparation, sample loading, and electrophoresis conditions to achieve optimal protein separation.
Prepare the resolving and stacking gels. The table below provides a basic recipe to make 1 gel (10% acrylamide). Make sure you optimize it based on the size of your protein of interest.
Larger proteins should have a lower percentage of acrylamide in the gel.
Remember to create the wells in your gel by carefully inserting a comb while the stacking gel is still wet.
| Acrylamide (40%) | 1.25mL | 0.25mL |
|
Separating buffer (4X) |
1.25mL | - |
| Stacking buffer (4X) | - | 0.625mL |
| Deionized water | 2.5mL | 1.625mL |
| 10% APS | 50μL | 25μL |
| TEMED | 5μL | 2.5μL |
| Total (mL) | 5mL | 2.5mL |
Only add the APS and TEMED when you are ready for the gel to set!
Once the gel is set, gently remove the comb. It's time to load equally across wells. Include a molecular weight ladder in one of the lanes.
Be careful not to touch the bottom of the wells with your pipette tip, as this can create distorted bands.
It helps to load your samples asymmetrically to help orient the gel during transfer.
Run the gel at a constant voltage (100-200v) until the desired separation is achieved.
Running times and voltages should be optimized, e.g., larger proteins will require a higher voltage for a longer period of time.
Once SDS-PAGE is complete, carefully pry open the apparatus with a gel knife and remove the gel.
Use an ice pack or perform the transfer in a cold room to prevent overheating.
Protein Transfer
Following electrophoresis, proteins must be transferred from the gel onto a membrane for detection. This transfer step is crucial to ensure that target proteins are accessible for antibody binding. There are two primary transfer methods: wet transfer, which is ideal for larger proteins, and semi-dry transfer, which is faster and suited for smaller proteins. Below, we outline the steps for each method and tips to ensure efficient protein transfer.
Choose your membrane type:
- PVDF has a higher binding capacity, so is best for low-expressed proteins.
- Nitrocellulose is ideal for proteins with low molecular weight.
For a fluorescent western blot, Ponceau staining is not recommended as it can cause high background fluorescence, even after washing.
If you are using PVDF, you must first soak the membrane in methanol for 30 seconds.
Soak the membrane in transfer buffer for 10 minutes to help equilibrate the gel.
Prepare the transfer sandwich in the transfer cassette according to the illustration below.
Make sure you gently remove air bubbles with a small roller or pipette.
Make sure you put the gel closest to the negative electrode and the membrane closest to the positive electrode.
Perform either a semi-dry or wet transfer.
Choose semi-dry transfer for a faster, easier transfer of small-volume or small-sized (<30kDa) proteins. Opt for wet transfer if you want to transfer a larger (>100kDa) target protein.
Semi-Dry Transfer:
- For this method, place the transfer sandwich horizontally between the two plate electrodes in the transfer apparatus.
- Apply a constant current (0.1 to 0.4 A) or voltage (10 to 24 V) for 10 minutes, or up to an hour.
Wet Transfer:
- For this method, cushion the transfer sandwich with pads, press them together with a support grid, and place them vertically in a tank filled with transfer buffer.
- Apply a constant current (30V or 1A) for one hour, or up to overnight.
(Optional) Confirm the success of the protein transfer using Ponceau.
Blocking and Antibody Incubation
Blocking and antibody incubation are key steps in Western blotting, ensuring specific binding of antibodies to the target protein while minimizing background noise. The choice of blocking buffer, antibody concentration, and incubation times can significantly impact the clarity and specificity of your results. Follow the steps below to optimize your antibody binding for high-quality detection.
Incubate the membrane in a blocking buffer for either one hour at room temperature, or overnight at 4°C with gentle rocking.
The blocking buffer can be either BSA, milk powder, casein, or Gelatin. For guidance on which to choose, see our FAQs.
Dilute the primary antibody to working concentration with blocking buffer and incubate the membrane in the solution (either with gentle rocking overnight at 4 °C, or 1 hour at room temperature).
Wash the membrane with wash buffer (either TBS, TBST, or PBS) for 10 minutes, three times.
Incubate the membrane with the diluted secondary antibody for one hour at room temperature.
Wash the membrane again (for 10 minutes, three times) to remove any unbound antibody.
Detection and Quantification
Once antibodies have bound to their target proteins, detection methods such as chemiluminescence, fluorescence, or colorimetric assays are used to visualize protein expression. Proper imaging and quantification help ensure that results are reproducible and provide meaningful biological insights. Below, we outline the key steps for detecting and analyzing your protein bands.
If using or chemiluminescent detection method, prepare the chemiluminescent substrate solution according to the manufacturer's instructions and incubate the blot in the substrate solution for 1-5 minutes.
Remove any excess substrate by dabbing the edge of the blot with tissue paper.
Place the blot in the imaging system tray or scanner.
Image your blot, adjusting exposure as needed.
Remove the membranes from the scan bed, and clean the image scanning bed.
To quantify, utilize image analysis software that supports band detection and quantification.
Troubleshooting & Optimization Tips for SDS-PAGE and Western Blot Protocols
Even the most well-planned experiments can encounter unexpected challenges. Common issues such as poor protein transfer, weak signals, and high background noise can significantly impact data quality and reproducibility.
Using high-quality buffers and reagents, combined with a well-optimized protocol, can minimize the risk of these common challenges. Below are some common issues encountered during the SDS page and western blot protocol, alongside some key strategies to help you troubleshoot and optimize your results.
My bands are faint or smeared
The problem:
This could be a result of the proteins degrading, potentially caused by issues with sample preparation and handling.
What to do:
- Keep samples on ice: Always work on ice throughout the lysis and sample preparation process to slow down protease activity.
- Use fresh inhibitors: Immediately add fresh protease and phosphatase inhibitors to your lysis buffer.
- Minimize handling time: Process samples quickly and keep them in a cold environment to further reduce degradation risk.
My bands are 'smiling' or distorted
The problem:
Curved or distorted bands ('smiling bands') may indicate uneven migration during electrophoresis.
What to do:
- Adjust the voltage: Smiling bands can indicate the voltage is too high, reduce the voltage and make sure you keep the gel cool during electrophoresis by keeping it on ice or in a cold room.
- Check the gel polymerization: Incomplete or uneven polymerization can lead to variations in gel density, causing bands to curve. Check your gel recipe to ensure the quantities are correct, and make sure the gel is covered entirely in buffer while setting.
My target bands are faint or missing
The problem:
If proteins do not transfer fully from the gel to the membrane, your target bands might be faint or missing entirely.
What to do:
- Confirm the transfer success: After transfer and before continuing the western blot protocol, check whether the proteins were transferred successfully with Ponceau S or Coomassie staining.
- Increase the transfer time or voltage: Adjust the transfer parameters by either extending the transfer duration or increasing the voltage (but be sure to keep everything cool).
- Adjust the buffer composition: Make sure the transfer buffer is freshly prepared and that its composition is optimal.
- Check assembly: Ensure that the transfer sandwich is assembled correctly without any air bubbles between the gel and membrane, and make sure that the transfer was performed in the right direction.
My bands are obscured and difficult to distinguish
The problem:
Excessive background noise, which can be caused by issues with blocking buffer, antibody concentrations, or washing, can make bands hard to see
What to do:
- Optimize blocking buffer: Experiment with different blocking agents to see which one best reduces non-specific binding.
- Increase wash times: Extend the washing steps to ensure that unbound antibodies are thoroughly removed.
- Review antibody dilutions: Ensure that the primary and secondary antibody concentrations are appropriate; too high a concentration can lead to non-specific binding.
All my bands have weak or no signalÂ
The problem:
A weak or absent signal may be due to several factors, such as insufficient antibody binding or problems with the transfer process.
What to do:
- Adjust antibody concentrations: Increase the concentration of the primary antibody, and increase the incubation time.
- Confirm proper transfer: Verify protein transfer using a reversible stain like Ponceau S.
- Check the amount of protein: It may be that not enough protein present so ensure that enough is loaded into each well.
Why Boston BioProducts?
Consistent buffer composition, ultrapure chemicals, and carefully formulated blocking agents can improve protein separation, enhance target detection, and reduce non-specific binding. Whether it's selecting the right lysis buffer for efficient protein extraction, using high-purity SDS sample buffers for proper denaturation, or employing optimized detection reagents for clear, high-sensitivity results, each component plays a crucial role in data quality.
By integrating Boston BioProducts' high-quality reagents into your SDS-PAGE and Western blotting protocol, you can achieve clearer, more consistent results, increasing the reliability and reproducibility of your protein analysis, and reducing troubleshooting time.
FAQs
Wet transfer (tank transfer) is highly efficient for large proteins and involves a buffer-filled chamber, taking 1-2 hours or overnight for best results. Semi-dry transfer uses less buffer and is faster (15-45 minutes), but it may be less effective for large or high-molecular-weight proteins
The percentage of acrylamide in your gel determines how well proteins of different sizes separate:
- Low percentage gels (4-8%): Best for resolving large proteins (>100 kDa).
- Medium percentage gels (10-12%): Suitable for most proteins (20-100 kDa).
- High percentage gels (15% or higher): Ideal for small proteins (<20 kDa).
Selecting the right blocking buffer is essential for reducing background noise and ensuring strong, specific antibody binding in your SDS-PAGE and western blot protocol. Which one you choose depends on your target protein, antibody type, and detection method.
- Non-Fat Powdered Milk: This is a cost-effective and widely used option for general Western blot applications. It works well with most primary and secondary antibodies but can interfere with phospho-specific antibodies due to its protein content.
- Bovine Serum Albumin: Ideal for phospho-specific and glycoprotein antibodies, as it lacks phosphoproteins that could cause background issues. It is also a good alternative when milk-based blocking buffers fail.
- Casein: A purified milk protein that provides effective blocking while minimizing cross-reactivity. This is good for fluorescent Western blots and when using certain monoclonal antibodies.
- Gelatin: A good option for reducing background in fluorescent and chemiluminescent detection. It works well with antibodies that show non-specific binding in milk or BSA.
Proper cell lysis is crucial for extracting proteins efficiently while minimizing degradation. The method varies depending on whether you are working with adherent cells or suspension cells.
For adherent cells:
- Aspirate PBS and add the ice-cold lysis buffer—you should add around 1mL per 107 cells.
- Incubate on ice for around 10 minutes, agitating occasionally.
- Collect the lysate and transfer to a microcentrifuge tube.
For suspension cultured cells:
- Centrifuge the cells at 2,500 x g for 10 minutes.
- Discard the supernatant, resuspend the pellet in ice-cold PBS, and centrifuge at 2,500 x g again for 10 minutes.
- Carefully resuspend the pellet in ice-cold lysis buffer (around 1 mL per 100 mg of wet cell pellet) and incubate for 10 minutes.
- Transfer to a microcentrifuge tube.
Before running SDS-PAGE, it's important to determine protein concentration to ensure equal loading. Common assays include: Bradford Assay, Bicinchoninic Acid (BCA) Assay, Lowry Assay and UV Absorbance at 280 nm.
BCA and Bradford assays are the most commonly used due to their ease and compatibility with common lab reagents - such as PBS and BSA. Bradford assays are great for rapid screening, high sensitivity, and cost-efficiency, whereas BCA should be your assay of choice for high throughput, consistency across protein types, or if your samples contain detergent.