Having trouble detecting phosphorylated HSL. Any tips for how to prevent de-phosphorylation events or detect phosphorylation?
If it were me, I would check literature for papers that successfully detected phospho-HSL and purchase the antibody they used. Follow the company’s instructions for use. Purchase phosphorylation inhibitors for use in your lysis buffer and add just before lysis As they are unstable in aqueous solutions.
if you are looking for stimulated p-HSL, cells should be serum deprived for a period of time before stimulus to see largest increase. Hope this helps.
Hello Molly Feighny
You may incorporate the following in your protocol.
1. You need to understand the mechanism of phosphorylation. If stimulation is required to phosphorylate the protein of interest, you should test different concentrations and perform a time-course experiment. This will help you to understand the extent to which the phosphorylation is transient and enable you to choose the appropriate time point for your experiment.
2. You must keep your phospho-protein intact. When you lyse your sample, endogenous phosphatases are released into the lysate which can rapidly dephosphorylate the protein of interest. To prevent this, you should add phosphatase inhibitors like Sodium fluoride (10mM) and Sodium orthovanadate (2mM) fresh to your lysis buffer just before you perform lysis. Protease inhibitors should also be added to prevent sample degradation. You should use the smallest volume of lysis buffer possible to concentrate your sample.
3. When detecting phosphoproteins, you should not use non-fat dry milk for blocking because milk contains the phospho-protein casein, which can potentially be detected on the membrane by anti-phospho antibodies, resulting in high background. Therefore, instead of milk consider switching to an alternative blocker such as BSA.
4. Avoid using phosphate-based buffers because the sodium phosphate contained in phosphate-based buffer like PBS can interact with anti-phospho antibodies, affecting the binding of the antibody to the target protein. When you must detect phosphoproteins by Western Blot, it is better to use Tris-based buffers like Tris-buffered saline with Tween-20 (TBST).
5. To detect phosphorylation on Western Blot, it is necessary to select antibodies which are highly specific for the phosphorylated protein state only.
6. Use PVDF membrane when you must detect phospho-protein on Western Blot because PVDF has a higher binding capacity than nitrocellulose, and so preferred for low abundance phosphorylated targets.
7. You should probe for total protein. Using an antibody that detects the total level of your target protein will enable you to determine the phosphorylated fraction relative to the total fraction. This will allow you to compare the impact of different treatments and will also provide an internal loading control. You may do this by multiplexing so that you can look at the target total protein as well as phospho-protein at the same time.
Best.
Prepare fresh lysis buffer and add total protease/phosphatase inhibitor (Thermo Catalog No.PI78441). Use phospho-specific mono antibody.
Use BSA (5%) for blocking and Ab dilution instead of skimmed milk. TBST for washing the membranes. Use enough lysate (>30ug). You should've no issues. Best, J
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