i injected immunized hemolymph of larvae into FPLC (fast protein liquid chromatography)
to separate peptides from each other.
every peak of protein separated in a falcon tube in 5 ml of Sodium chloride buffer.
next step is lypholization , after samples were lypholized , protein powder plus sodium chloride crystal is precipitated .
should i do desalting for protein to have a pure protein or can i use it as a whole for inhibition assay fro bacteria??
If the samples are equivalent in their salt content you may be able to do the experiment with suitable controls (buffer no protein, buffer + known inhibitor) . If your purification process involved a gradient elution (for example) the different fractions could contain much different salt concentrations and might be best to exchange them into identical buffers for your test. A reverse phase elutuion might be completely normalized by lyo, as the organic would be removed, but an ion exchange elution could be quite different. What was the separation method?
Edward Michelini Thanx for replaying, the separation method was Ion Exchange elution , and that is true the samples are not equivalent in the salt concentration.
so what is the proper method should i use to remove sodium chloride crystal , with the knowledge that i have very small samples in volume ( 40 micro liter) and i will use sodium acetate as a buffer.
It is important to evaluate the activity of a pure compound. You could use RP-HPLC fractions would be monitored by mass spectrometry. Electrospray ionization mass spectrometry (ESI-MS) is useful to directly determine the exact mass of the antimicrobial peptide in RP-HPLC fractions. Characteristic charge state patterns of the protein or peptide compound upon ESI-MS analyses will reveal further structural information about the the compound. Because ESI-MS is highly sensitive towards the presence of salt, analyses of ion exchange-HPLC fractions requires elimination of inorganic salts, either by the use of volatile buffers or reverse phase cartridges for each separate fraction. In the case of cation exchange-HPLC, salts are required for protein elution. Because several antimicrobial peptides are salt sensitive, there will be a principal risk of getting false-negative results. In some cases, it is possible to overcome this problem by using unconventional, pragmatic conditions for cation exchange-HPLC using volatile buffers such as ammonia salts at an acidic pH. For details see: Book Antimicrobial Peptides: Methods and Protocols
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