1X Protein Lysis Buffer
|G032||5.0 ml (5 x 1.0ml)|
abm’s 1X Protein Lysis Buffer is optimized to release proteins of interest from cells and tissues for use in Western Blot experiments. The buffer includes anionic denaturing detergent sodium dodecyl sulfate (SDS) and bromophenol blue, which allows quick preparation of denatured and reduced protein samples from cells. A brief sonication and heating at 100°C for 5 minute will allow the samples from direct loading onto SDS-PAGE gel. abm’s 1X Protein Lysis Buffer provides an easy and efficient way for SDS-PAGE and Western Blot sample preparation.
SDS-PAGE, Western Blot
|Composition||SDS, Tris HCl, glycerol, bromophenol blue, DTT.|
Pre-thaw and thoroughly mix the 1X Protein Lysis Buffer before use.
|Unit quantity||5.0 ml (5 x 1.0ml)|
Store at -20°C upon receiving.
|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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