Hi everyone, I transfected HEK cells with a protein that is intracellularly expressed through flow. Then I isolated the protein using RIPA buffer protocol, however, I accident added IP buffer instead of RIPA and now I do the know if it’s worth going through the whole western blot process.
For more context, I only know the protein shows intracellular expression through flow but don’t know exactly where it’s expressed. I looked up and ThermoFisher said IP is less harsh than RIPA so I don’t know if it collects everything or not.
Any advice is appreciated!
The problem is that different people use the name RIPA (radioimmuno assay precipitation buffer) and also immune precipitation buffer for very different things. RIPA buffer, for example, is a Tris-buffered salt solution with various additives (https://en.wikipedia.org/wiki/Radioimmunoprecipitation_assay_buffer). For IP buffer, the concentration of detergents is (OK, should be) titrated for each protocol for the best compromise between cleanest blot and maximum sensitivity. Without careful comparison of the composition of the buffers you were (or should have been) using, your question is unanswerable.
One thing, however, you should think about: Science is about repeatability. If you switch buffer, you start a new series of experiments. Is that worth it, or would it be easer to just make a new preparation.
Hello Emma Pham
I feel it would not be worth going through the process of Western Blot using IP lysis buffer.
RIPA buffer is a denaturing buffer and is more stringent buffer because of the presence of SDS and sodium deoxycholate. As RIPA does not maintain native protein conformation, proteins that are difficult to release, such as nuclear proteins, can be released with denaturing buffer like RIPA. The buffer’s harsh properties are best suited for hard to solubilize proteins, which is why RIPA is the preferred choice.
On the other hand, IP lysis buffer is a modified RIPA buffer formulation which is without SDS. This moderate-strength lysis buffer effectively solubilizes cellular proteins but does not disrupt protein complexes like the RIPA lysis buffer. It contains NP-40, which is milder, non-ionic detergent. It is good at solubilizing membrane proteins and for isolating cytoplasmic proteins. Proteins retain their native state in the presence of this detergent and protein-protein interactions are preserved. Such gentle formulation helps maintain protein complexes for co-immunoprecipitation.
When you lyse your cells with RIPA buffer, all proteins within compartments, including nuclear and mitochondrial proteins will be released. This is due to the combination of harsh denaturing, ionic detergents (sodium deoxycholate and SDS) and the milder, non-ionic detergent (NP-40).
So, going further is not worth, if you are using IP lysis buffer instead of RIPA. Moreover, you do not know where the protein is expressed intracellularly.
Best.
