Hi Anil and Rashmi,

Please let me  clarify. The FA is conjugated/saponified with KOH at 65-70C. That complex is added to pre-warmed BSA at 37C. On occasion, the FA-BSA complexes precipitate out or look cloudy anyway. A short incubation at 50-55 usually fixed that. This was an optional step that I used only if need be. Rashmi, I did it too all my samples if I did it to one. Anil, BSA is very robust and in this case, it's just a carrier for a label (in my case it was an alkyne or azido-FA for palmitoylation or myristoylation assays). 

I hope that helps.

I would think it is necessary because FFA (free fatty acids) do have detergent like properties and could damage your cells. Also they are poorly soluble in aqueous media. These are part of the reasons why they are transported in blood bound to albumin.

In general, it will help the uptake. Typically, free fatty acids are toxic to cells. Depending on what you are looking at, it might be best to saponify your fatty acid and then mix with fatty acid free BSA. Again, it all depends on what you are trying to do. 

In fact most protocols suggest conjugating both PA and FFA-free BSA in a particular ratio to get the desired concentration of PA complexed to BSA... However, you can try dissolving palmitic acid in isopropyl alcohol at a stock concentration of 40 - 80 mM (you can keep it at -20ºC) and then adding it to DMEM containing 1% FFA-free BSA to get the final experimental concentration. This works at least to treat hepatocytes with palmitic acid.

You can take a look at this paper, where they dissolve palmitic acid like that:  "Free Fatty Acids Induce JNK-dependent Hepatocyte Lipoapoptosis" Harmeet Malhi, Steven F. Bronk, Nathan W. Werneburg and Gregory J. Gores; JBC, 2006.

I used step dilution (from PA in 100% ethanol) to get PA to the correct concentration (0.4mM) in DMEM. This method worked for me. I found the PA-BSA conjugation to be really tricky with either the PA precipitating out or the BSA denaturing at high temperature.  

Hi, I followed a protocol that palmitic acid is conjugated into 5% BSA. I set the BSA stirred at 40 degree celsius while keeping the PA at 70. After I finished conjugating the PA into the BSA, the solution was not transparent but a little opaque. Does it seem right? Should I set the BSA at higher temperature, eg. 55oC according to some people? (I follow the Seahorse company not to keep the BSA over 40)

I have seen it be opaque and work fine for labeling palmitoylated or myristoylated proteins. I have also placed the BSA at 55C or higher for about 5 mins and then place it back to 37C. That also worked. We've switched to adding the palmitate to 20% FAFBSA and then add to cells so that the BSA is 1% final in cell culture. But we also saponify our FAs with a 20% molar excess of KOH. Forming the salt and then binding to BSA increases the uptake of the FA and decreases toxicity of using a freeFA. 

Dale - Do you have a saponification and conjugation protocol you would be willing to share? I need to feed my cells some 13C-palmitin but I can't seem to find a decent saponification protocol to convert my palmitin to a salt prior to BSA conjugation.

C16:0 was dissolved in 100% ethanol (250 mM or 500 mM) then mixed with BSA in a 10:1 molar ratio in 0.25% FBS/DMEM or 0.25% FBS/RPMI medium. The mixture was sonicated

in a water bath for 15 min followed by incubation on a shaker in an oven at 55 C for 15 min. The solubilized C16:0 was ltered through a 0.22 u m lter before use.

http://www.ncbi.nlm.nih.gov/pubmed/22766885

Hey Bob, not sure what happened, but I responded yesterday right away from my phone. Anyway, you can check my pubs and it should be in the Yap et al JLR paper or Martin et al FASEBJ 2008. If not, I can send you something more detailed. Basically, we start with a 20% molar excess of KOH to the FA. Heat that and then add to prewarmed 37C BSA (we have used both 2x and 20x BSA). This was primarily to look at fatty acylation of proteins, but I think it should be used when adding fatty acids since most of them are toxic at high concentrations. They activate the JNK pathway. If you are using radioactive palmitate for palmitoylation, I recommend using Click chemistry. You can buy the reagents way cheaper from Cayman drugs. Also, don't follow the invitrogen instructions. They never work. Follow our paper. Good luck. 

Hello,

I am using 10%BSA-conjugated palmitate to treat my cells. I prepare 5mM palmitate-BSA stock, by adding heated palmitate solution (dissolved in 0.1N NaOH at 70c) to warm BSA at 55c. My question is, need I boil THIS 5mM STOCK SOLUTION to 55c for 10-15 minutes everytime before giving treatment (as suggested in few protocols) and why  ?

Thanks,

Rashmi

I only prewarm my BSA to 37. I might heat it to 55 if it precipitated. 

Thanks Dale ! Will you share detail protocol for BSA-Palm preparation and treatment  ? Thanks again !

Hi Rashmi, I actually already shared it above, but I think now that we are on the second page of answers you can't see it unless you go back a page. 

 Dale,

Sorry if it is already discussed in the paper you shared. I was wondering how effective would complexing be if its done at 50 degree? BSA is supposed denatured at this temperature. Thanks. 

Hi Dale, Why don't you treat the FA-BSA solution same way as the BSA control solution before giving the treatment ? Wouldn't that be necessary for comparison between control and FA ?

Hi Anil and Rashmi,

Please let me  clarify. The FA is conjugated/saponified with KOH at 65-70C. That complex is added to pre-warmed BSA at 37C. On occasion, the FA-BSA complexes precipitate out or look cloudy anyway. A short incubation at 50-55 usually fixed that. This was an optional step that I used only if need be. Rashmi, I did it too all my samples if I did it to one. Anil, BSA is very robust and in this case, it's just a carrier for a label (in my case it was an alkyne or azido-FA for palmitoylation or myristoylation assays). 

I hope that helps.

Hello,

This thread was very informative and helpful but I have a question. Does anyone know exactly how the palmitic acid is conjugated to BSA? Is it a chemical or electrostatic conjugation? I am curious how the molecules interact to conjugate allowing for increased uptake into the cells.

Thank you!

It should by non-covalent interactions, likely hydrophobic. BSA is a carrier in serum, and should be acting in the same manner here. 

https://fscimage.fishersci.com/cmsassets/downloads/segment/Scientific/pdf/Cell_Culture/Application_Notes/Lipids_Media.pdf 

I found a paper stating that, salt bridge is formed between the carboxyl group of FA and basic amino acids of BSA