We recently encountered a weird difference between two extraction methods that should yield the same results, and we could not find an explanation:
1. Cell lysis was performed using a hypotonic buffer with 0.5% NP-40 followed by centrifugation at 16,000g for 15min (in a cooled centrifuge).
2. Cell lysis was performed using a needle (25G) followed by centrifugation at 16,000g for 15min (cell lysis was verified).
In both methods we get no detection of our protein in the supernatant (cytosolic fraction). However, when we dissolve the pellet in triton-based buffer our protein is extracted only in method #1. We know it is in the pellet in method #2 since it can be extracted using SDS.
We could not explain why triton only extracts our protein in method #1, so maybe someone here would have some idea.
Thank you in advance.
Hi,
As I understood, you don’t have any detergent in method #2. In my opinion, you might be lysing the cells but, without detergent, membranes might stay intact and form micelle-like structures, in which you hydrophobic protein is protected from triton.
Dear Sachar, I have no specific data on this. But it looks like you are dealing with a hydrophobic type of protein, since it can be extracted by triton in method 1 and by SDS in the pellet fraction from method 2. Hydrophobic proteins require usually stronger detergents to isolate them. Is your protein of interest a hydrophobic type?
Dear Johan, thank you for your answer. We know it is hydrophobic, since it is not in the supernatant. We were wondering how can we explain the differences in triton-solubility with the two extraction methods.
Hi,
As I understood, you don’t have any detergent in method #2. In my opinion, you might be lysing the cells but, without detergent, membranes might stay intact and form micelle-like structures, in which you hydrophobic protein is protected from triton.
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