What are the most effective ways to prepare your samples for Western blotting?

1 Collecting samples

The first step to prepare your samples for Western blotting is to collect them properly. Depending on the type and source of your samples, you may need to use different methods and tools to obtain and preserve them. For example, if you are working with animal or human tissues, you may need to use surgical tools, scissors, or biopsy punches to cut and weigh them. If you are working with cells, you may need to use trypsin, centrifugation, or pipetting to detach and collect them. In any case, you should always handle your samples gently and quickly, and avoid any contamination or degradation.

2 Storing samples

The next step to prepare your samples for Western blotting is to store them appropriately. Depending on the stability and sensitivity of your proteins of interest, you may need to use different temperatures and additives to prevent their loss or modification. For example, if you are working with proteins that are prone to proteolysis, oxidation, or phosphorylation, you may need to use protease inhibitors, antioxidants, or phosphatase inhibitors in your storage buffer. If you are working with proteins that are unstable or volatile, you may need to use low temperatures, such as -80°C or liquid nitrogen, to freeze them. In any case, you should always label your samples clearly and avoid any repeated freeze-thaw cycles.

3 Lysing samples

The third step to prepare your samples for Western blotting is to lyse them effectively. Depending on the composition and complexity of your samples, you may need to use different methods and reagents to break down the membranes and release the proteins. For example, if you are working with tissues, you may need to use mechanical methods, such as homogenization, sonication, or bead beating, to disrupt the tissue structure. If you are working with cells, you may need to use chemical methods, such as detergents, salts, or chaotropic agents, to solubilize the cell membranes. In any case, you should always choose a lysis method and buffer that are compatible with your proteins of interest and your downstream applications.

5 Loading samples

The final step to prepare your samples for Western blotting is to load them properly. Depending on the size and charge of your proteins of interest, you may need to use different volumes and additives to separate them by electrophoresis. For example, if you are working with large or acidic proteins, you may need to use low volumes and high concentrations of reducing agents, such as dithiothreitol (DTT) or beta-mercaptoethanol (BME), to denature and reduce them. If you are working with small or basic proteins, you may need to use high volumes and low concentrations of loading buffer, such as sodium dodecyl sulfate (SDS) or bromophenol blue, to coat and visualize them. In any case, you should always use a suitable molecular weight marker and a positive control to compare and verify your samples.

6 Here’s what else to consider

This is a space to share examples, stories, or insights that don’t fit into any of the previous sections. What else would you like to add?