Most protocols suggest using a lysis buffer, spinning, and then adding Laemmli buffer to the supernatant. However, I have heard that it is easier to simply lyse the cells with 1x Laemmli buffer, heat at 100C for 10 minutes, quench on ice, quick spin, then load. Is this a better protocol for westerns? It certainly seems easier. Apparently, this method prevents the loss of nuclear/golgi proteins because the lysate is not spun at a high speed. The only problem seems that this protocol can produce a very sticky lysate that is hard to load. I have heard that this is due to lipids.
What is your lysis protocol for a western blot? Why must lysis buffer be used on ice? Are nuclear/golgi proteins lost in the pellet during high speed spins?
Thanks!
I never use straight laemmli buffer to prepare cells because it is generally not compatible with protein assays so the samples can't be properly quantified. People who use this typically plate a constant number of cells and then collect the cells at some later point in laemmli buffer to try to match the "quantity" of cells. But, there can be many experimental conditions that will alter the proliferation of cells - if that happens then the quantity of protein loaded onto the SDS-PAGE is not matched. If cells are first collected in a solubilzation/lysis buffer, then the supernatants can be used for a protein assay so equal amounts of protein are loaded on the gel. Also, if you are not specifically looking for nuclear or mitochondrial proteins, you will get a better Western if they are spun out of your sample before loading.
David: It depends on how much dilution you were loading to the wells. We are doing it the same as yours, but use a very diluted and liquid-like sample thanks to our exceptional sensitive home-made primary Ab-HRP. Check out our paper (attached) for details . Good luck.
If you find it useful, please "Recommend" as a way of saying thanks :=)
I never use straight laemmli buffer to prepare cells because it is generally not compatible with protein assays so the samples can't be properly quantified. People who use this typically plate a constant number of cells and then collect the cells at some later point in laemmli buffer to try to match the "quantity" of cells. But, there can be many experimental conditions that will alter the proliferation of cells - if that happens then the quantity of protein loaded onto the SDS-PAGE is not matched. If cells are first collected in a solubilzation/lysis buffer, then the supernatants can be used for a protein assay so equal amounts of protein are loaded on the gel. Also, if you are not specifically looking for nuclear or mitochondrial proteins, you will get a better Western if they are spun out of your sample before loading.
I generally prefer using a lysis buffer like RIPA buffer for isolation and later process the samples for Western. Using Laemmli buffer straightaway would also give you good Western results, but I've personally felt that the former is better if you plan on storing your protein samples for a longer time. Also, the points that Beedle raised about compatibility issues and mismatch in conditions are important. As mentioned, when I used Laemmli buffer, it was for an experiment that had constant number of cells.
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