You’re probably reading this post because you are experiencing ladder migration during SDS-PAGE. The good news is: we’re here to help. At Azure Biosystems, we are dedicated to helping researchers quickly obtain consistent, reliable data. Just take a look at the 3,000 publications in our resume to see for yourself. Generating reliable data depends largely on troubleshooting when things go wrong. In this blog post, I am going to address this question from ResearchGate, “Why does the ladder separate, but the samples are not migrating or separating?” Keep reading for my three top tips for foolproof SDS-PAGE.
AZURE EXPERT TIP: Double-check the Sample Buffer
Proteins occur in complex tertiary and quaternary structures. For proper migration with SDS-PAGE, the proteins must be denatured so the structure does not affect their ability to migrate through the gel. To denature proteins, sample buffer should contain SDS. Additionally, the samples should be heated for further denaturation.
In addition to the denaturation, proteins also need to be negatively charged in order to move through the gel during electrophoresis. The SDS used in the sample buffer is responsible for this step. SDS essentially coats proteins in a negative charge.
Ensure your sample buffer contains all of the ingredients needed, in the right concentrations, and that nothing has expired. If there is an issue with migration, it is probably best to make fresh sample buffer to be safe.
AZURE EXPERT TIP: Try heating things up
Proteins occur in complex tertiary and quaternary structures. For proper migration with SDS-PAGE, the proteins must be denatured so the structure does not affect their ability to migrate through the gel. To denature proteins, sample buffer should contain SDS. Additionally, the samples should be heated for further denaturation.
In addition to the denaturation, proteins also need to be negatively charged in order to move through the gel during electrophoresis. The SDS used in the sample buffer is responsible for this step. SDS essentially coats proteins in a negative charge.
Ensure your sample buffer contains all of the ingredients needed, in the right concentrations, and that nothing has expired. If there is an issue with migration, it is probably best to make fresh sample buffer to be safe.
The final step in protein denaturation is boiling the sample. While technically not required for all samples, it is good practice to include this as part of the sample preparation process. Heating adds an extra level of breaking protein bonds to linearize the proteins as much as possible.
Ensure the sample preparation protocol includes a heating step. The heating step is as follows:
1. Simply put the samples in a heat block at 95°C, for 2-5 minutes.
2. After heating, place the samples directly on ice until ready to load into the gel. This step prevents renaturation of proteins that can occur if the samples are allowed to cool to room temperature naturally.
AZURE EXPERT TIP: Use a reducing agent
Some protein structures contain disulfide bonds. Since SDS does not disrupt disulfide bonds, a reducing agent may be added to the sample buffer to take care of this issue. The two common reducing agents used in SDS-PAGE are DTT and 𝛃-mercaptoethanol. Ensure these are added to the sample buffer to completely denature the proteins. If one is added, try making new buffer and exchanging the reducing agent used. DTT is stronger than 𝛃-mercaptoethanol so the reducing agent used could make a difference.
How does SDS-PAGE separate proteins?
After reading these steps are you finding you still don’t have a basic understanding of how SDS-PAGE works? Let me explain the process to you.
Using electrophoresis, proteins migrate through a polyacrylamide gel. Because of being denatured and then coated in a negative charge, the proteins will migrate and separate based on size. To determine the size of the proteins, a molecular weight marker, or protein ladder, is used. Often, these ladders are pre-stained so you can visually detect the progress of the protein separation.
Unfortunately, the ladder migrating and separating appropriately does not guarantee that the proteins in your sample have done the same. Most of the time when there are migration issues with SDS-PAGE, we would consider a number of factors such as buffers and gel quality; however, in this case, the molecular marker is an indicator that those things are working appropriately. This leaves the question of how are the protein samples different from the protein ladder?
The protein ladder is purchased and undergoes quality control to ensure that it is indeed prepared properly and ready for use in SDS-PAGE. With your protein samples however, you have to prepare them yourself. The goal of the sample preparation is denaturation and adding a negative charge to allow the proteins to travel. If either of these are messed up, then protein separation would not occur with SDS-PAGE, while the protein ladder would continue to migrate.
We hope you find these troubleshooting steps to be fairly easy to implement. With these simple changes, your bands will be separating in no time. LabXChange does a good job of explaining the purpose of proteins in SDS-PAGE using this graphic– check it out.
For more information about how Azure Biosystems can help you with analyzing proteins, check out our reagents and equipment here. If you are still experiencing issues, use the form on this page to ask our experts for help. See you next time!
