I have to treat my cells with a-synuclein protein monomers, oligomers and fibrils but for sterilizing we have used 0.22 syringe filter for monomers before fibrillation induction. Now we found that filtering decrease protein concentration which can cause low fibrillation efficiency. Therefore, I would like to use another method for sterilizing proteins without using syringe filters.
You can also pre-treat the filter by passing a 0.1% BSA solution through the filter. This will reduce/block the non-specific binding of your protein to the filter. After the BSA, rinse the filter with PBS and you should be ready to go
Dear Amir,
You may sterilize your protein by radiation. though there are chances of some protein alteration but i guess in your case it can help. Use radiation along with the use of antibiotics. I hope your culture would not get contamination.
Best luck..
Radiation might damage your protein, so could entail some risks. Probably better to use thimerosal to maintain sterility.
You can also pre-treat the filter by passing a 0.1% BSA solution through the filter. This will reduce/block the non-specific binding of your protein to the filter. After the BSA, rinse the filter with PBS and you should be ready to go
Use a 0.45 µm filter. Your protein should go through, leaving bugs behind.
How long will take your experiments? I mean you treat the cells with the protein and then which is incubation time and so on?
You can try using alcohol - water mixture to sterilise your protein. While ethanol may cause some aggregation (Biochemistry. 2003 Mar 11;42(9):2720-30; https://www.researchgate.net/publication/10876634_Conformational_behavior_and_aggregation_of_alpha-synuclein_in_organic_solvents_modeling_the_effects_of_membranes) that can be adjusted and may be reversed.
Article Conformational Behavior and Aggregation of α-Synuclein in Or...
How are you making your fibrils is you are synthesizing the peptide just keep everything sterile and work in the hood so there is no need to filter...
I agree with two of the respondents suggestions regarding filter selection and pretreatment. Specifically, you should flush the filter first with a low concentration BSA solution dissolved with the buffer your protein in suspended in (or just the buffer alone if you are concerned about trace BSA in your subsequent reactions) and try various different filter-membrane polymers (e.g. PVDF, PFTE, PES) to determine which one has the lowest binding affinity to your protein.
Another way to pretreat membranes is with ovalbumin or poly-l-lysine, then rinse with PBS.
Most membrane filters have a nominal pore size achieved through a meshwork of polymer fibers. You might try Nuclepore filters (polycarbonate) with a smooth surface and etched, neat holes; and Whatman makes PTFE (Teflon) filters in various pore sizes (not pleasant to work with, IMHO).
Do you achieve the same loss in protein concentration via centrifugation? That means a better disruption/solubilization method could get more of the monomer into solution. Have you tried sonication?
Fabio Formiggini : We usually treat cells 24h with protein then maybe with do another treatment for 24h as well.
Janice Bramham : We did filteration several time with same sample and we observed atypical decrease in OD 280nm! I think, if there was that problem, aggregate forms should be removed just the first time.
I agree with Elizabeth Gordon. It depends a lot 1) on the source of your peptide 2) on the time your cells will be in contact with the peptide. Point 1) gives you the expected bacterial (or fungi, virus,...) load, i.e. how many microorganisms you can expect to be in your peptide preparation. Consider that the procedures to synthesize peptides and/or to purify them are very well controlled (and often too strong for microorganisms to survive), therefore you should expect close to zero contamination. Point 2) gives you the probability that the few microorganisms present (if any) will have the chance to a) adapt to the new conditions b) divide c) overcome cell defense and d) eventually kill your cells. Longer time means higher probability.
You could even test your preparation for microbial presence, using for example a bit of your peptide and incubating it in suitable broth (even enriched) and/or medium where your cells grow. Very likely, you will be surprised to see after several days, no grow at all of microorganisms.
Of course, when you resuspend the peptide is mandatory to use sterile water and/or sterile buffer (these can be 0.22um filter sterilized without any problem, but take care about preservatives which are included in the filter itself, if any, wash the filter twice with water before filtering the solution you will use: preservatives could affect your cells).
So, consider very well if you really need to filter your precious peptide.
Good luck
Fabio
Following from Janice Bramham, I have tried determining the change in protein concentration of a 1% BSA in PBS solution before and after 0.22um syringe filtration. In my case, the concentration did not change, thereby indicating that almost all the BSA passed through the filter. It might indeed be a problem with protein aggregation. I used a Nanodrop to measure the absorbance at 280nm. You may also opt to use any UV-Vis Spectrophotometer instrument.
Hello
I also have a very similar problem and would appreciate your feedback. I have a nano-sized biological sample in very lsmall volume (200 ul media), I believe I can't get them to the size that can pass the syringe filter because when I try to filter, can't press on the syringe and it pops out. Is there a way to reduce aggregates of protein to filter them ?
Thanks
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