Western Blot Ladder Transferred but Not Protein: Challenges and Solutions

When it comes to western blotting, achieving superior resolution is crucial for separating peptides and small proteins with a molecular weight less than 10 kDa. One of the key challenges in this process is ensuring that the protein marker or samples are loaded correctly, particularly when dealing with very small proteins. To overcome this issue, we recommend using a loading tip to minimize the problem of insufficient stacking.

In addition to loading techniques, the choice of gel composition and running conditions can also impact the resolution of western blotting results. For example, Tricine gels are ideal for separating peptides and small proteins, while gels with higher than 15% acrylamide may be less effective due to technical difficulties in preparation.

Another important consideration is the stacking layer thickness, which should be less than 5 mm from the well bottom to the top of the separation layer. If the stacking layer is too long, it can lead to insufficient stacking and ambiguous results. Furthermore, gels with higher than 15% acrylamide may not provide satisfactory resolution due to issues such as uneven polymerization, bubble formation, and inaccurate protein size estimation.

To address these challenges, we suggest using a gel with 15% acrylamide in the Tris-Glycine system, which is capable of well-separating small proteins ranging from 5 to 10 kDa. Additionally, it is important to calibrate the molecular weight against interested proteins by comparing the migration pattern of pre-stained markers with unstained protein markers.

In conclusion, western blotting can be a complex and challenging process, particularly when dealing with very small proteins. By understanding the challenges and solutions outlined above, researchers can optimize their western blotting protocols to achieve superior resolution and accurate results.

Western blot ladder transferred but not protein