The goal of this step is to break your cells while trying to maintain protein’s integrity as much as possible. So, before starting, make sure to get some ice. You’ll keep everything cold, including buffers.
First step: Wash cells with PBS
If working with suspension cells, this step is very easy. Just spin-down cells and wash with cold PBS trying to avoid to break them. Let’s say 300-500g will be ok (Yes, this will translate to a different number of RPM depending on the centrifuge). After the last wash, try to remove all the PBS. As completely removing it is impossible, try to get the same final volume in all samples.
If working with adherent (that is attached) cells, wash cells with cold PBS (to prevent cells detaching, use PBS containing calcium and magnesium) and make sure to remove all the PBS from the cells before adding the lysis buffer. If there is any left PBS, it will affect the next steps. Once all PBS is removed, proceed immediately to next step (we don’t want that cells get dry).
Second step: Add lysis buffer
Yes, is easy to say, but there are several lysis buffers. So…
¿what’s the best lysis buffer?
Here there are the 4 most common lysis buffers, from softer to stronger:
1. Hypotonic Buffer
Tris-HCl pH7.5 10mM
Hypotonic Buffer is the softer buffer listed. Is useful if you are interested in a cytoplasmic protein as won’t disrupt most of the nucleus.
2. NP-40 (also known as igepal/Nonidet)
Tris-HCl pH8 50mM
3. Triton X-100
Tris-HCl, pH8 50mM
Triton X-100 1%
NP-40 and Triton X-100 are very similar, being Triton X-100 buffer slightly stronger. They are useful for most of the situations.
4. RIPA BUFFER (Radioimmunoprecipitation assay buffer)
Tris-HCl pH=7.5 50mM
Triton X-100 1%
Caution, both SDS and Deoxycholate are toxic reagents
This is the most stringent buffer in the list, and can be even more by raising NaCl content to 200mM or even 300mM. Choose this one if you are looking for transcription factors or other nuclear proteins. Is highly recommended to add a sonication step to the procedure in order to break the DNA, otherwise some proteins could be lost with the sticky DNA.
Third step: Add loading buffer and Boil.
First, spin down the lysate at 4ºC (Max speed) and take the supernatant (discard cell debris). Then, add Loading Buffer.
Loading buffer choice depends on the kind of gel that will be used in the next steps. Usually boiling time ranges between 2 and 10 minutes. (Some proteins are sensitive to heat, take it into account).