Protease Inhibition Cocktail with EDTA
|Description||Protease Inhibitor Cocktail is used to prevent the digestion of proteins that occurs after cell lysis takes place. The presence of EDTA ensures greater inhibition of metalloproteases.|
|Composition||100mM PMSF, 100mM EDTA, 2mM Bestatin, 0.3mM Pepstatin A, and 0.3mM E-64|
|Protocol Overview||1mL of the Protesase Inhibitor Cocktail is recommended for the inhibition of protease activity form 100mL of cell lysate at a density of 10^8 cells per mL.|
However, since not all organisms contain the same level of endogenous proteases, it is up to the researcher to optimize the concentration for their experiments.
|Shipping Conditions||As this item contains PMSF, please operate carefully. PMSF is toxic and causes irritation to the eyes and skin. Wear eye and hand protection and proper lab attire when handling the item.|
|Unit quantity||1.0 ml|
|Storage Condition||Store cocktail at -20°C. Cocktail is dissolved in DMSO containing little deionized water. The cocktail may form crystals. If there are crystals, place it at RT for 5 minutes. The cocktail is stable for a year.|
|I would like to know under what circumstance I could have no signal?|
Here are some suggestions and how you could resolve the problem: 1. The primary antibody and the secondary antibody are not compatible. Use secondary antibody that was raised against the species in which the primary was raised (e.g primary is raised in rabbit, use anti-rabbit secondary). 2. Not enough primary or secondary antibody is bound to the protein of interest. Use more concentrated antibody. Incubate longer (e.g. overnight) at 4ºC. 3. Cross-reaction between blocking agent and primary or secondary antibody. Use a mild detergent such as Tween20 or switch blocking reagent (i.e. commonly used blocking reagentsare milk, BSA, serum or gelatin). 4. The primary antibody does not recognize the protein in the species being tested. Check the datasheet or perform a ClustalW alignment to ensure your antibody should react with the target protein; Run the recommended positive control. 5. Insufficient antigen. Load at least 20-30 ug protein per lane; Use protease inhibitors; Run the recommended positive control. 6. The protein of interest is not abundantly present in the tissue. Use an enrichment step to maximize the signal (e.g. prepare nuclear lysates for a nuclear protein, etc.). 7. Poor transfer of protein to membrane. Check the transfer with a reversible stain such as Ponceau S; check that the transfer was not performed the wrong way; if using PVDF membrane make sure you pre-soak the membrane in MeOH then in transfer buffer. 8. Excessive washing of the membrane. Do not over wash the membrane. 9. Too much blocking does not allow you to visualize your protein of interest. Instead of using 5% milk in the antibody buffers try removing the milk or using 0.5%; Switch blocking reagents or block for less time. 10. Over-use of the primary antibody. Use fresh antibody as the effective concentration is lowered upon each re-use. 11. Secondary antibody inhibited by sodium azide. Do not use sodium azide together with HRP-conjugated antibodies. 12. Detection kit is old and substrate is inactive. Use fresh substrate.
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