western blot ladder transferred but no proteins

Is everything in the sandwich properly hydrated? Were there no bubbles, including on the hydrophobic membrane?.Did you handle the membrane properly? That is, did you handle it with gloves, and ensure that there are no pressed marks if using laser capture?.To minimize your number of stupid mistakes, always ask yourself the following questions before you start ANY Western blot transfer (no matter how routine): In the laboratory it is common to be multitasking, overworked and distracted-all things that lead to stupid mistakes. While we like to think that we are above stupid mistakes, we are not. It is faster to make a new gel than to do a new sample preparation AND a new gel. If you see any of these problems, chuck your gel and start over. Before you use your gel, gently tilt it to ensure that it is completely set up. If your gel can be seen shrinking from the edge, it is drying out. These imperfections will impair protein migration. Properly made wells should be uniform and rinsed. To avoid losing your samples to a bad gel, always examine your gel closely before use. A Bad GelĪfter hours of performing experiments and preparing samples, you better make sure your samples are loaded onto a pristine gel! However, even if you do everything right-you followed the recipes carefully, minded you pH, used fresh reagents, meticulously cleaned your plates-things can still go wrong with your gel. An optimize protocol will maximize your protein-of-interest’s solubility and stability, which will allow you to better visualize your protein and reliably interpret your Western blot data. Proper sample preparation is an empirical process involving the comparison of detergent extraction versus whole cell lysate, salt concentrations, buffer types, inhibitors (to prevent degradation and loss of post-translational events), reducing agents and heating times.

While labs often have their own sample preparation methods, consider optimizing your own process. Bad protein samples due to improper or sub-optimal protein preparation can result in poor Western blots.

Proper protein extraction and sample preparation is critical. While some of these mistakes are perplexing, others are just plain dumb but none of them have to happen to you. Sadly this is usually due to mistakes on the experimenter’s part. As bitter experience has likely taught you, not all Western blots are pretty.

Western blot ladder transferred but no proteins