5X Protein Loading Buffer
|Description||Simplify and standardize your sample loading with abm’s 5X Protein Loading Buffer. This pre-mixed and ready-to-use reducing sample buffer is provided in a convenient 5X formulation. Mix one part sample buffer with four parts protein sample, heat denature and then load the sample in the gel wells. Visualize progress of the electrophoresis run by monitoring the location of the blue dye front. Protein Loading Buffer may be used in denaturing gels of all kinds and are compatible with coomassie dye and silver staining techniques, as well as Western blotting procedures.|
|Composition||Tris-HCl pH 6.8, 10% w/v SDS, 250 mM DTT, 0.03% w/v bromophenol blue, and 50% v/v glycerol|
|Unit quantity||3.0 ml|
Store at -20°C for long-term storage or at 4°C for short-term storage.
|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.
|Is this loading buffer adequate for reducing SDS-PAGE? Or will I need to add another reducing agent to my samples?|
Yes, this 5X loading buffer also acts as a reducing agent for denaturing proteins.
- Sharma, V et al. "Registered report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation" eLIFE 5:e11414 (2016). DOI: 10.7554/eLife.11414. Application: Western Blot.
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