Problem	Possible reasons	Potential Solutions
Underdeveloped neuronal processes	1. Neurons were not healthy 2. Culture used earlier than DIV-14	1. Plate neurons at adequate density and ensure the quality of culture media 2. Allow neurons in culture for DIV-14 or more
Failure of LD induction	KLH45 was degraded	Reconstitute fresh KLH45, store at appropriate temperature in small aliquots and avoid freeze-thaw cycle
Incomplete lysis of neurons	Pressure of nitrogen in cell disruption vessel was low	Increase the pressure in nitrogen vessel
Low count of LDs	1. Incomplete lysis of cells 2. Gradients layers of high volume 3. Loss during LD collection	1. Ensure complete lysis of neurons 2. Use smaller tube for small samples 3. Tube slicer can be used
Aggregation of LDs	Electrolyte in the solution was low	1. Check the concentration and pH of buffers 2. Additional floatation step in low concentration of glycerol, 0.1-1 M NaCl or 100 mM Na2CO3 (pH 11.5)35
Disrupted LD membrane	Homogenization was too harsh	Reduce the nitrogen pressure
Contamination of membrane	1. Wide diameter tube used 2. Density gradient was messy	1. Preferably use tubes of narrow diameter for prep. with small number of neurons 2. Use smooth pipette with wide orifice tips on a sturdy platform
High phospholipid in TLC	Membrane contamination	Follow the above steps and ensure the quality of LDs before lipid extraction
Smearing of protein bands in western blot	1. Proteins were degraded 2. Incomplete extraction of lipids during protein precipitation	1. Add protease inhibitors to all MEPS buffers and set-up the gradient on ice 2. Increase the centrifugation speed to separate the lipid phase from proteins