Preparing Palmitate:BSA solution for treatment of cells? - (Mar/28/2012 )
Hello everyone!
I'm beginning a new project that involves treatment of a particular cell line with the free fatty acid, palmitate/palmitic acid(PA).
I'm currently looking for protocols to make up the solution but cannot seem to find any that go into any great detail on how to prepare the solution and was hoping to find a reliable method by someone who works regularly with fatty acids.
Most protocols that I have found share the same principal of initially making stock solutions of both PA and FFA-free BSA, followed by mixing each solution in a particular ratio to get the desired concentration of PA complexed to BSA.
One aspect of the protocol that I'm unsure of is how to best prepare the PA stock solution as once you open your new vial of fatty acid, and expose it to the air, it becomes oxidized.Would it then be best to make up a stock solution at once using all of the PA in the container followed by aliquoting out multiple tubes containing the stock solutions, or, to aliquot the powder into several tubes and make up the solution fresh each time you wish to do your treatments?
Thanks!
We have had palmitic acid on our fridge shelf for years and it seems to be fine. If in doubt, just dissolve it in 100% DMSO (it will be tough to get it to a concentration above 50 mM). Palmitic acid is very stable in solution, unlike unsaturated fatty acids so a stock kept at -20 C should be no problem.
We regularly treat bacteria with fatty acids an commonly use media containing 10 mg/ml FFA-free BSA and the desired concentration of fatty acid.
The research in our lab is focused on the biochemistry of FA + phospholipids so any more questions on the handling of fatty acids, let me know.
Bluefunk on Sun Apr 1 18:21:08 2012 said:
We have had palmitic acid on our fridge shelf for years and it seems to be fine. If in doubt, just dissolve it in 100% DMSO (it will be tough to get it to a concentration above 50 mM). Palmitic acid is very stable in solution, unlike unsaturated fatty acids so a stock kept at -20 C should be no problem.
We regularly treat bacteria with fatty acids an commonly use media containing 10 mg/ml FFA-free BSA and the desired concentration of fatty acid.
The research in our lab is focused on the biochemistry of FA + phospholipids so any more questions on the handling of fatty acids, let me know.
Could you tell me in more detail how you make up your stock solution in DMSO and what stock concentration you use in the lab? Do you know if it is difficult to get the PA into solution? I'm going to try make it up in DMSO this evening as I could not get it into solution using 0.1M NaOH.
Thank you for your help
Of course, the working concentration depends on the purpose of the experiment. 16:0 fatty acid is very insoluble in water, I think the limit of solubility is around 50 micromolar. Making a stock in DMSO couldn't be easier; calculate the mass you need for a 50 mM solution, add appropriate volume of 100% DMSO and heat at 37 degrees + vortex until it dissolves. It is one of the more insoluble fatty acids so is indeed a little tricky to get into solution but heating in DMSO should fix that. PA is very stable, as there are no double bonds oxidation should not be an issue. The only functional group on the FA is the terminal carboxylic acid and that only reacts under specific conditions.
Any more questions, let me know
Palmitic acid will also dissolve in 100% ethanol quite easily.
Bluefunk on Mon Apr 2 14:07:18 2012 said:
Of course, the working concentration depends on the purpose of the experiment. 16:0 fatty acid is very insoluble in water, I think the limit of solubility is around 50 micromolar. Making a stock in DMSO couldn't be easier; calculate the mass you need for a 50 mM solution, add appropriate volume of 100% DMSO and heat at 37 degrees + vortex until it dissolves. It is one of the more insoluble fatty acids so is indeed a little tricky to get into solution but heating in DMSO should fix that. PA is very stable, as there are no double bonds oxidation should not be an issue. The only functional group on the FA is the terminal carboxylic acid and that only reacts under specific conditions.
Any more questions, let me know
Thanks for all the information however I do have another quick question. Do you sterile filter your PA solution? I have received mixed reports from people saying that PA can/cannot be filtered and wanted to know what you do in your particular lab?
I am asking this as I am not getting the expected toxicity in my cells treated with PA. I'm attempting to repeat an experiment published in a particular paper and I'm to figure out whether or not the PA is the problem.
Thanks again by the way. I really appreciate your advice.
We never filter sterilize our fatty acids that are dissolved in 100% DMSO and have never experienced contamination in our broth/agar plates containing FA. The solvent itself is very inhospitable for bacteria. The same applies for FA dissolved in methanol/ethanol - sterilization is simply not necessary.
No problem, once again feel free to ask anything else
Hi there, I have got a question related to this post. I am trying to treat mouse pancreatic islets with palmitate these days. I dissolve Palmitic acid (not palmitate sodium) in 100% ethanol, vortex it, heat at 60C for a few mins and vortex again to dissolve it completely. I then couple it to 1% BSA (fatty acid free-from Roche: Cat# 10775835001) such that the final Palmitate concentration is 0.5 mM. BSA solution is prepared in complete CMRL medium containing 10% FCS. I use this medium containing Palmitate and BSA to treat islets for 4 days. I am seeing a brownish dust/fine precipitate in my culture medium after 3-4 days that is killing all the islets. It seems like BSA is coming out of solution in this long term culture. I have made sure that the culture samples are not contaminated. Further, this brown dust is also seen in control samples containing BSA only. So, adding palmitate is not the reason here. I have used complete CMRL without BSA many times and never had this kind of problem. I have also tried serum free medium+BSA but had same problem. Can anyone help me with this issue please?