We all know how sensitive and time consuming the process of preparing lysate samples for western blots is. You spend days, if not weeks, meticulously growing, treating, and harvesting cells praying that you don’t mess anything up. Finally you reach that last step: lysing the cells. Right as you are squirting the lysis buffer onto the cells you see it collecting in the corner of the plate……. cloudy goop. You ask yourself “Is that right? How do I get that into the tube?” and you are left questioning your super scientist skills for fear of sabotaging your experiment yet once again. Fear not, for Team-Phospho is here to help!
Let’s talk “goop”. Not the most scientific word one should use to describe lysate samples, but is all too common in the preparation of lysate samples. You wonder…. Is my protein in the goop? YES! Did all of my cells get lysed? Most likely yes, if you used the correct detergent. In most cases people use the standard RIPA buffer, which contains 1% Triton or NP-40 as a detergent, for lysing cells. Both of these detergents are nonionic and are not optimal for solubilizing membranes or transmembrane proteins. SDS, which is a great ionic detergent for solubilizing membranes and transmembrane proteins, is sometimes added to RIPA buffer. However, most RIPA buffers only contain 0.1% SDS, which is not enough to solubilize those membranes. We recommend at least 1% SDS in your choice of lysis buffer to see complete solubilization of membranes. Additionally, heating the 1% SDS lysis buffer to 95˚C before lysing will further enhance the process so that no goop is present and scraping is not needed.
Coincidently, lysis buffer with 1% SDS will appear goopy by itself when placed on ice. Once samples that have been successfully lysed with 1% SDS they can be left a room temperature for a short period of time and do not need to be placed on ice. If there is a little goop, try sonicating the sample in 5 second pulses at a low frequency. This will not negatively affect the lysate, it will only further assist in solubilizing the membranes and transmembrane proteins. The final step in solubilizing the goop is to heat fix the lysate samples at 95˚C for 5 minutes. Once you have followed these few steps after harvesting your samples they will be ready for the next epic endeavor…..western blotting!
Take home message:
-Make sure you are using an ionic detergent when lysing adherent cells. We recommend 1% SDS.
-Look after lysing: Look at your plate after lysing and collecting the sample. If cells are still present, lyse again with hot 1% SDS lysis buffer (95˚C).
-Don’t ice lysate samples that contain 1% SDS, they will be goopy. If you made this mistake just heat and sonicate the samples again, the goop is gone!
-Samples should only be left a room temperature if used the same day, for long term storage we recommend storing the lysates at -20C. Just remember to heat and sonicate before pipetting the lysate sample due to goop.
-Sonicate your sample in short intervals at low frequency.
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