Try these 5 simple tips to make your next western blotting experience a breeze!!!
Contributed by Kameron Simpson
1. Use a Lysis Buffer Containing 1% SDS
Use lysis buffer containing 1% SDS when preparing whole cell lysates to ensure membrane and other hard to solubilize proteins, even synaptic junction proteins, are completely solubilized.
- 1% SDS lyses everything, preserving proteins from protease and phosphatase enzymes. Additional cocktail inhibitors are not necessary when 1% SDS is used.
2. Use Fresh BME In Your Sample Buffer
When preparing lysate samples use fresh BME in 4X sample buffer to ensure samples are properly reduced and denatured.
- BME reduces the sulfhydryl groups in proteins and is highly volatile. We recommend adding fresh BME to 4X sample buffer just prior to adding to your samples.
3. Maximize the number of antibodies you can test on one blot
When testing multiple antibodies using the same lysate, try using a stacking layer with one large trough instead of multiple lanes to maximize the number of strips available for testing.
- Once the gel has been transferred to a blot, you can use a razor blade and ruler to cut the blot into strips of equal widths. In most cases, twice as many strips can be cut from one large trough blot than can be cut from a blot with multiple lanes. The strips can then be incubated in a tray with narrow wells.
4. Visualize the proteins on your blots
Stain your membrane with Ponceau S after transfer to confirm successful protein transfer and to determine the exact alignment of lanes and placement of the proteins on the gel.
- Ponceau S will show you where protein has/ has not transferred onto the membrane and allow you to visualize any areas where bubbles or other factors may have affected the transfer of protein from the gel to the membrane.
Make sure to collect excess Ponceau S for reuse and to thoroughly rinse membranes in dH2O after staining to remove any remaining excess Ponceau S. Ponceau S staining is reversible and will not interfere with antibody labeling of the membrane.
5. Try A Dilution Curve
Optimal primary antibody dilutions should be determined experimentally using a dilution curve. A dilution that is too low will give a signal that is oversaturated. A dilution that is too high will give a signal below the detection threshold. Finding the “Goldilocks” zone where the signal is detectable, but not saturated is the goal.
- Experimental protocols and imaging systems vary from lab to lab. Trying a dilution curve with the primary antibody allows you to determine the best dilution to use in your system.
Take Home Messages:
- A lysis buffer containing 1% SDS should be used when preparing whole cell lysates to ensures all proteins are completely solubilized.
- Always use fresh BME in 4X sample buffer to ensure samples are properly reduced and denatured.
- When running a gel try using a stacking layer with one large trough instead of multiple lanes to maximize the number of antibodies that can be tested with the same lysate.
- Stain your membrane with Ponceau S after transfer to visualize protein transfer and protein alignment on the membrane.
- Optimal primary antibody dilutions should be determined experimentally using a dilution curve due to differences in experimental protocols and imaging systems that often vary from lab to lab.