Foolproof Guide to SDS-Page Ladder Migration

6 minute read

JOIN THE AZURE INSIDER CLUB

For the latest publications, promotions, and news on upcoming products sent weekly to your inbox

ASK A SCIENTIST

Got a question? Let us help! Describe the problem you’re having and one of our experts will reach out.

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. Generating reliable data depends largely on troubleshooting when things go wrong. In this blog post, I’m going to address the question:

“Why does the ladder migrate and separate as expected during SDS-PAGE, but the experimental samples do not?”

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. Simply put the samples in a heat block at 95°C, for 2-5 minutes. 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.

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!

Check out recent blog posts

Shopping cart
There are no products in the cart!
Continue shopping