Hey!

There could be several reasons why your protein bands are not running fully on the SDS-PAGE gel, i will try to put it broader way

1. Insufficient denaturation: SDS-PAGE relies on the denaturation of proteins by SDS and β-mercaptoethanol to ensure uniform charge-to-mass ratios. If your samples are not properly denatured, the proteins may not migrate correctly through the gel.

2. Protein aggregation: If the proteins in your samples are aggregated or have formed large complexes, they may not penetrate the gel effectively, leading to incomplete migration.

3. Incorrect gel composition: The percentage of acrylamide in the gel affects the resolution of different protein sizes. A 15% running gel is suitable for separating small proteins, but if your protein of interest is larger, you may need to use a lower percentage gel.

4. Poor sample loading: Uneven or overloaded sample application can cause distortion of the gel matrix and hinder protein migration. Ensure that you are loading an appropriate amount of protein in each well.

5. Improper running conditions: Running the gel at too high a voltage or for too long can cause excessive heating, leading to distorted protein bands or even denaturation of proteins. Try reducing the voltage or running time to optimize protein migration.

6. Gel degradation: If the gel has been stored improperly or for an extended period, it may degrade, resulting in poor resolution and incomplete protein migration.

To troubleshoot your SDS-PAGE experiment, you may consider adjusting denaturation conditions, optimizing sample loading, verifying gel composition, and ensuring proper running conditions. Additionally, it may be helpful to run a molecular weight marker alongside your samples to assess gel performance and confirm the expected migration pattern of proteins.

What I do:

I generally run at 70V for 20 mins (till it crosses stacking gel) and then 150 for 50min. I have attached the gel composition that i use. i hope that helps!

Regarding First time SDS, Running SDS-PAGE for the first time can indeed be challenging, but with practice and experience, you'll become more adept at troubleshooting and optimizing your experiments. Stick with it, and don't be discouraged by initial setbacks. Familiarizing yourself with the gel composition and adjusting running conditions based on your specific samples is crucial. Over time, you'll develop a better understanding of how different factors, such as voltage, running time, and gel composition, influence protein migration and band resolution. Remember to keep experimenting and refining your techniques, and don't hesitate to seek advice or guidance from experienced colleagues or online resources. With perseverance and patience, you'll master SDS-PAGE and achieve consistent and reliable results.

Thank you for your detailed answer!

The first sample in the gel is the marker so i would rule out insufficient denaturation, protein aggregation and incorrect gel composition because not even the marker ran. About running conditions, I think that might be the problem, because I noticed there’s a power outage.