- Use A Lysis Buffer Containing 1% SDS
Use a 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.
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.
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.
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.
Proteins of different molecular references transfer from gel to membrane at different speeds and efficiencies. Proteins with larger molecular references will take longer to transfer than proteins with lower molecular references. Try staining your gel with coomassie after the transfer to confirm that the proteins of interest have migrated out of the gel. Remember to also consider protein loss across the membrane. By adding a second membrane behind the first, you can see to what extent the proteins have blown through. Process both membranes with your antibody. Use data from such experiments to optimize transfer time for the specific protein of interest.
When working with phospho-tyrosine antibodies, try blocking and incubating with 3% and 1% BSA respectively instead of milk. Milk often contains high concentration of phospho-tyrosine and thus can give an abnormally high background if used as a blocking agent with phospho-tyrosine antibodies.
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.
Antibodies are sensitive and must be handled and stored with care to ensure they remain active and efficient in binding their target proteins. If you don’t plan on using all of the antibody right away, freeze it in aliquots (volume dependent on application) at -20C or lower when they are not being used and keep your “working aliquot” stored at 4C.
- Aliquots from antibodies formulated with 50% glycerol in their buffer can be repeatedly taken directly from the stock vial stored at -20C without freeze/thawing due to the glycerol content.
Demystifying SDS-PAGE: The science behind all of those bubbles
Western blot protocol
Blog: 1% SDS is the buffer of choice for most western blots
Lysate preparation protocol
Blog: The Truth About Goopy Lysate
Blog: Antibody Dilution Recommendations – What You Need to Know!