Hello, I work on FABPs, I have a question about delipidation of FABPs after purification. I would appreciate if anyone could help me. Thank you in advance.
To delipidate a protein using Lipidex-5000, you can follow these steps:
1. Prepare a solution of Lipidex-5000 in a suitable buffer (e.g., 50 mM Tris-HCl, pH 7.5) according to the manufacturer's instructions.
2. Mix the purified FABP with the Lipidex-5000 solution and incubate for a suitable period of time (e.g., 30 minutes) at room temperature with gentle agitation.
3. Centrifuge the mixture at a suitable speed (e.g., 10,000 x g) for a suitable time (e.g., 5 minutes) to separate the Lipidex-5000 beads from the delipidated protein.
4. Collect the supernatant, which should contain the delipidated FABP, and analyze its purity and lipid content by SDS-PAGE and lipid staining methods.
Note that the effectiveness of Lipidex-5000 in removing lipids from FABPs may vary depending on the specific protein and lipid composition. Therefore, it is important to optimize the delipidation conditions for each protein to achieve the desired level of delipidation.
@Lamia Fatima Thank you Fatima for the detailed answer. Just one thing, in which ratio should I mix protein and lipidex 500?
@sanam Mohammadzadeh
The ratio of protein to Lipidex-5000 may vary depending on the specific protein and lipid composition, as well as the desired level of delipidation. However, a commonly used ratio is 1:5 (protein:Lipidex-5000, w/w) (Yan et al., 2014). It is important to note that this ratio should be optimized for each protein to achieve the desired level of delipidation.
Reference:
Yan, N., Fan, C., Chen, Y., Hu, Y., & Yang, Y. (2014). One-step purification of recombinant human heart-type fatty acid-binding protein using ion-exchange monolithic chromatography. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 957, 71–75. https://doi.org/10.1016/j.jchromb.2014.03.015
However, the optimal ratio may need to be determined empirically for each specific protein and lipid composition. It is recommended to perform a pilot experiment using different ratios of protein to Lipidex-5000 to determine the most effective ratio for delipidation.
@Lamia Fatima
I really appreciate your help Fatima! Thank you so much.
Dear Fatima Lamia Fatima ,
Hi, I have a question about buffer used for prepering lipidex solution: my protien is in 20mM HEPES ph 7.7 , 1mMTECEP and 100 mM NaCl. I was wondered why many protocol for delipidation used phosphate buffer. I cannot find why. Does it matter which buffer you use for prepering lipidex solution?
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