you can prepare solution in DMSO or BSA as per your concentration needed
Ethanol, methanol and THF you can use as a solvent to prepare solution.
Hi Wang,
Though this is an old post, found few difficulties to get a proper protocol to make Palmitic acid. This one down is pretty handy.
1. Dissolve 0.1282g of PA in 1 mL (500 mM) of 100% ethanol and heat at 70°C
2. Make 10% BSA in cell culture media (DMEM, KSF, etc.), filter and incubate at 37°C
3. Add 10 uL of dissolved PA into 990 uL of 10% BSA
4. Vortex and incubate at 55°C for 15 min
5. Repeat the step 4
6. Use 10 uL of ethanol in 990 uL of 10% BSA to make control
7. Store the samples in -20°C
8. Incubate at 55°C for 15 min before treat cells
9. The final PA concentration in BSA is 5 mM
10. PA to BSA ratio is 3.2:1
Reference:
Kim JY, Lee HJ, Lee SJ, Jung YH, Yoo DY, Hwang IK, Seong JK, Ryu JM, and Han HJ. Palmitic Acid-BSA enhances Amyloid-beta production through GPR40-mediated dual pathways in neuronal cells: Involvement of the Akt/mTOR/HIF-1alpha and Akt/NF-kappaB pathways. Sci Rep 7: 4335, 2017.10.1038/s41598-017-04175-w.
Cheers,
Susara
Susara Madduma Hewage
Hi i have tried the given protocol, it worked. Though i wanted to increase the PA stock conc to higher value. Can u tell me if i want to make 50mM or 10 mM, what % of bsa is required. Since I have tried making 10mM stock of PA in same way, it didnt worked out
Hello Anant,
I’m glad to hear that the protocol worked..!
The most critical step in making PA is adding BSA to the proper ratio. We know that plasma albumin possesses about seven binding sites for fatty acids with moderate to high affinity, enhancing the concentration of fatty acids by several magnitude orders [1]. Therefore, I would say we can maintain PA: BSA ratio of less than 7:1.
Increased BSA concentrations trigger protein overload and stress out the cells. Therefore, it’s always good to use 10% BSA or less to prepare PA stock solutions.
I tried to make 10mM of PA stock but it didn’t completely dissolve PA in BSA. However, I could make an 8mM stock in 10% BSA, and it works without an issue. Just give a try for this recipe.
Note: I tried to store this in -20C. However, the PA insult reduces over time once they were treated to cells. So, I would make it fresh each time.
Cheers..!
1. van der VUSSE, G.J., Albumin as fatty acid transporter. Drug metabolism and pharmacokinetics, 2009. 24(4): p. 300-307.
@Susara Madduma Hewage Hi, thanks for sharing your protocal, that is very helpful. I still a question.
for this part : 1. Dissolve 0.1282g of PA in 1 mL (500 mM) of 100% ethanol and heat at 70°C
2. Make 10% BSA in cell culture media (DMEM, KSF, etc.), filter and incubate at 37°C
3. Add 10 uL of dissolved PA into 990 uL of 10% BSA
4. Vortex and incubate at 55°C for 15 min
5. Repeat the step 4
6. Use 10 uL of ethanol in 990 uL of 10% BSA to make control
7. Store the samples in -20°C
8. Incubate at 55°C for 15 min before treat cells
9. The final PA concentration in BSA is 5 mM
10. PA to BSA ratio is 3.2:1
after step 4,does it still need a water sonicator for 15 min at 55°C? we don not have a water sonicator in our lab. thank you very much.
Susara Madduma Hewage
Hi, thanks for sharing your protocal, that is very helpful. I still a question.
for this part : 1. Dissolve 0.1282g of PA in 1 mL (500 mM) of 100% ethanol and heat at 70°C
2. Make 10% BSA in cell culture media (DMEM, KSF, etc.), filter and incubate at 37°C
3. Add 10 uL of dissolved PA into 990 uL of 10% BSA
4. Vortex and incubate at 55°C for 15 min
5. Repeat the step 4
6. Use 10 uL of ethanol in 990 uL of 10% BSA to make control
7. Store the samples in -20°C
8. Incubate at 55°C for 15 min before treat cells
9. The final PA concentration in BSA is 5 mM
10. PA to BSA ratio is 3.2:1
after step 4,does it still need a water sonicator for 15 min at 55°C? we don not have a water sonicator in our lab. thank you very much.
Hi
The processing protocols are well explanined in the following paper:
https://doi.org/10.1007/s12161-021-02203-0
I hope this helps!
@Susara Madduma Hewage hi..all these times my palmitate was working fine but now it has stopped working. Though the conjugated solution looks clear as earlier. But it is not inducing death or protein expression in my cells as earlier. Any idea?
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