There are five different signs that indicate that there are problems with SDS-PAGE sample preparation: sample leaking out of the well during or after loading, samples clumping and not migrating, you aren’t seeing bands, you’re seeing too many bands, or you’re getting smeared bands.
SDS-PAGE sample preparation issues:
- Samples leaking out of the well during or after loading
- Samples clumping in the well and not migrating properly
- No bands are visible after staining the gel following electrophoresis
- Too many bands in your gel after protein electrophoresis
- Smeared bands in your gel
In this article, we will discuss the issues that commonly occur and for each of these situations, the troubleshooting steps might solve the problem.
Article table of contents:
Samples leaking out of the well during or after loading
Samples clumping in the wells and bands not migrating properly
No bands are visible after staining the gel following electrophoresis
Too many bands in your gel after protein electrophoresis
General good practices for SDS-PAGE
Protein samples leaking out of the well during or after loading
Seeing distorted and smeared bands during protein electrophoresis can be due to samples leaking out of the well, which is caused by sample overload, uneven loading, incorrect buffers, or air bubbles in the wells during loading. Proper loading helps prevent issues and leads to better results.
Figure 1. Example of how sample leakage would appear during protein electrophoresis.
First possible explanation: The loading buffer may not have enough concentration of glycerol. Glycerol in your protein sample helps them sink down inside the well during loading.
Troubleshooting suggestion: Check for glycerol concentration in your loading buffer. You might need to increase glycerol concentration.
Second possible explanation: You might need to improve your gel loading approach. Loading samples in a polyacrylamide gel might not be as easy as loading an agarose gel for DNA electrophoresis. Below are some suggestions to keep in mind.
Troubleshooting suggestion: If there are air bubbles inside the well while you load that well, the sample would spill out during loading. The way around this is to first take a little bit of running buffer and rinse the well with it before loading your actual sample. This ensures that all air bubbles escape the well before you load your sample – this is an effective way to prevent samples from spilling out of the well during or after loading.
Another common issue, especially with people new to acrylamide gels, is that they tend to overfill the wells during loading. This potentially can lead to samples spilling out.
As a general rule, you would not want to load the well more than a maximum of 3/4 of its capacity.
Also, try to load all wells with equal volume.
Protein samples clumping in the wells and bands not migrating properly
Improper band migration and sample clumping in the well during protein electrophoresis can result from loading too much protein in the wells, high salt or detergent concentration, or protein aggregation or precipitation in the wells.
Figure 2. Illustration of what improper migration would look like.
First possible explanation: Too much protein was loaded in the wells.
Troubleshooting suggestion: Check the concentration of proteins in your samples. A good practice is to load 10 µg of protein per well.
If too much protein is loaded in the wells, band resolution will also usually be poor.
Second possible explanation: Protein aggregation or precipitation in the wells.
Troubleshooting suggestion: Ensure the solubility of your proteins in sample lysate before loading them into the gel. In this case, take extra care during the extraction of your sample proteins by doing proper homogenization.
One way of doing this is by sonicating your sample source (cell culture, bacterial culture etc.) adequately followed by centrifugation to remove cell debris.
Another suggestion is to add DTT or BME in your lysis solution. These chemicals reduce protein aggregation by breaking secondary structures.
Heating your lysate also aids in getting your sample proteins into their primary structure and reduces aggregation.
If your sample proteins are hydrophobic, there are higher chances of their aggregation. For this, consider adding 4-8M urea in your lysate solution before loading.
DTT (Dithiothreitol) (> 99% pure) Protease free
Catalog ID | Size | Pricing | |
---|---|---|---|
DTT10 | 10 g | $ 59.00 | |
DTT25 | 25 g | $ 129.00 | |
DTT50 | 50 g | $ 229.00 | |
DTT100 | 100 g | $ 369.00 | |
DTT500 | 500 g | $ 1,645.00 |
Description
Dithiothreitol (DTT) is the common name of the more popular of the two Cleland’s Reagents (the other being Dithioerythritol or DTE). DTT is a powerful reducing agent that forms a stable six-membered ring with an internal disulfide bond which is resistant to oxidation. DTT is often used for the following: reducing the disulfide bridge of the cross-linker N,N′-bis(acryloyl) cystamine to break apart the matrix of a polyacrylamide gel, the reduction of disulfide bonds in proteins, the prevention of those bonds from forming between cysteine residues and lastly, DTT is often used to reduce thiolated DNA in order to minimize dimerization.
DTT is a nearly 7-fold stronger reduction agent than βME (β-mercaptoethanol) and has a less offensive odor and is less toxic.
Product Specifications
Catalog ID | DTT |
---|---|
CAS # | 27565-41-9 / 3483-12-3 |
MW | 154.25 g/mol |
Grade | MOLECULAR BIOLOGY GRADE |
Storage/Handling | Store desiccated at -20°C. |
No bands are visible after staining the gel following electrophoresis
Not seeing bands in your protein gel after electrophoresis could be due to sample loading issues, or that the proteins in your sample were not adequately charged with a negative charge.
Identifying the cause is crucial in troubleshooting and optimizing the experimental conditions to achieve reliable and accurate results.
Figure 3. Illustration of faint bands compared to sharp bands. Lane one contains aprotein ladder with crisp bands. Lane 2 is an example of faint to almost no bandsin a protein gel. Lane 3 is an example of clear protein bands. Lane 4 shows faintbands that can be still seen.
First possible explanation: Not enough protein was loaded into the wells.
Troubleshooting suggestion: Check the concentration of proteins in the samples you ran on the gel. A good practice is to load 10 µg of protein per well.
Second possible explanation: The proteins in your sample were not adequately negatively charged. Without enough of a negative charge on the proteins, they won’t migrate properly to the positive electrode and you won’t see proper bands.
Troubleshooting suggestion: Ensure that proteins in your sample have enough SDS. SDS confers an overall negative charge to the proteins that enables them to migrate to the positive electrode during electrophoresis.
Too many bands in your gel after protein electrophoresis
Too many bands in your protein gel can be due to sample degradation. This may occur because of the presence of proteases or phosphatases that can break down the sample and cause the multiple band effect.
Figure 4. Illustration of expected bands in lane 2 as well as too many protein bands due to degradation in lane 3 (ladder in lane 1).
Possible explanation: Proteins in your samples might have degraded.
Troubleshooting suggestion: Add protease and phosphatase cocktail inhibitors during protein extraction and storing. Repeated freezing and thawing also leads to protein degradation. So, a good practice is to aliquot your samples and store them at -800C until ready for loading into the gel.
Smeared bands in your gel
Smeared bands in a protein gel can be due to sample overloading, uneven sample distribution, or high salt concentration in your lysate. Overloading the gel can cause the bands to spread and blur, while uneven loading may result in uneven migration and smearing.
Figure 5. Illustrates smeared bands on a protein gel.
First possible explanation: Concentration of the salts in your lysate may be too high.
Troubleshooting suggestion: Dialyzing your sample prior to loading it onto the gel might help. Desalting columns also helps get rid of excess salts.
Another alternative would be to precipitate the proteins in your sample with trichloroacetic acid and then resuspending it in a buffer with a lower salt concentration.
Second possible explanation: Too much protein loaded in the wells
Troubleshooting suggestion: Check concentration of proteins in your samples. A good practice is to load 10 µg of protein per well.
General good practices for SDS-PAGE
Here are some things you might need to keep in mind while preparing and loading your samples in a polyacrylamide gel:
- Carefully load samples into the wells. Do not puncture the wells, eliminate air bubbles, and do not overload the wells.
- You will want to keep the protein content loaded across wells as uniform as possible. Also, try to load equal volumes in every well.
- Check that your loading buffer is properly made; without enough glycerol in your loading buffer, your samples might not sink into the well and can leak out.
- Samples need to be properly prepared with requisite chemicals and heating, as necessary. You should be careful that proteins in your samples are not degraded; use protease and phosphatase inhibitors as required, for this purpose.
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Description
Due to the optimized concentration of the various inhibitors, the ProBlock™ Gold shows excellent inhibition of protease activities and is therefore suitable for the protection of proteins during purification from animal tissues, plant tissues, yeast and bacteria. ProBlock™ Gold contains both irreversible and reversible protease inhibitors to inhibit serine, cysteine and other proteases. An separate bottle of EDTA is provided to inhibit metalloproteases.
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ProBlock™ Gold 2D Protease Inhibitor Cocktail (EDTA Free) [100X]
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Description
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ProBlock™ Gold 2D contains both irreversible and reversible protease inhibitors and inhibits serine, cysteine and metalloproteases etc. Due to the optimized concentration of the various inhibitors, the ProBlock™ Gold 2D shows excellent inhibition of protease activities and is therefore suitable for the protection of proteins extracted from animal cells / tissues, plants, yeast and bacteria etc.
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ProBlock™ Gold Extra Strength Protease Inhibitor Cocktail
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Description
GoldBio’s new ProBlock™ Gold Extra Strength Protease Inhibitor Cocktail is specifically formulated for the most demanding protein purification applications. The optimized concentrations of the various inhibitors in our Extra Strength cocktail deliver an enhanced dose of protease inhibition for the toughest research experiments. ProBlock™ Gold Extra Strength contains both irreversible and reversible protease inhibitors to inhibit serine, cysteine and other proteases.
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ProBlock™ Protease Inhibitor Cocktail -100, Plus EDTA
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ProBlock™-100, Plus EDTA is designed for large volumes of extraction/ lysis buffers and is supplied as 10 vials /kit, with each vial suitable for 100 ml.
ProBlock™-100, Plus EDTA protease inhibitor cocktail contains optimized concentrations of various protease inhibitors, which provide excellent inhibition of protease activities during protein purification. ProBlock™ is a superior general protease inhibitor cocktail that is suitable for purification from mammalian, plant, bacteria and yeast samples. The cocktail contains both irreversible and reversible protease inhibitors to inhibit serine, cysteine and other proteases. EDTA is packed separately for inhibiting metalloproteases. At 1X concentration, ProBlock™ inhibits over 90% of protease activities (e.g. Mouse Pancreas Extract 0.5 mg/ml protein).
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Description
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Description
ProBlock™ Gold mammalian protease inhibitor cocktail contains optimized concentrations of various mammalian protease inhibitors, which provide excellent inhibition of protease activities during protein purification from mammalian cells and tissues. The cocktail contains both reversible and irreversible protease inhibitors to inhibit serine, cysteine and specific inhibitors for mammalian proteases, such as aminopeptidases. A 1X concentration of ProBlock™ Gold Mammalian inhibits over 90% of protease activities.
Some proteins require divalent cations (Ca2+, Mg2+ or Mn2+) for their biological activity and the presence of chelating agents may be detrimental to the protein’s activity. Chelators, such as EDTA, inhibit the purification of proteins by immobilized metal affinity chromatography.
ProBlock™ Gold Mammalian is supplied with additional, optional EDTA and 1,10-Phenanthroline monohydrate which are provided for optimal inhibition of metalloproteases and may be added in the extraction buffer or lysate as needed.
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Description
ProBlock™ Gold Plant protease inhibitor cocktail contains optimized concentrations of various plant protease inhibitors, which provide excellent inhibition of protease activities during protein purification from plant. The cocktail contains both reversible and irreversible protease inhibitors to inhibit serine, cysteine, metalloproteases and specific inhibitors for plant proteases, such as aspartic proteases and aminopeptidases. A 1X concentration oft ProBlock™ Gold Plant inhibits over 90% of protease activities.
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