User:Vijay/Trouble Shooting/western: Difference between revisions
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*Membrane protein problem | *Membrane protein western blot problem | ||
If none of the above solves the problem and if you are working with the membrane protein then you can try the following... | If none of the above solves the problem and if you are working with the membrane protein then you can try the following... | ||
#Solubilize the membrane fraction using appropriate detergents | #Solubilize the membrane fraction using appropriate detergents |
Latest revision as of 14:06, 6 July 2007
Just Suggestions
- Reduce protein load in the gel (If you get too many/unwanted banding, in addition to your band)
- Increase protein load in the gel (If you do not see your band)
- Use higher percentage gel
- Wash properly (If too much of background)
- Check on the blocking step (If too much of background)
- Reduce or increase the concentration of the primary/secondary antibody (If you get excess or no signal for your band)
- Reduce or increase the exposure time after developing the film (If you get poor/strong signal)
- Try peptide blocking, if using a cocktail peptide (to minimize cross reactivity of the antibodies)
- Membrane protein western blot problem
If none of the above solves the problem and if you are working with the membrane protein then you can try the following...
- Solubilize the membrane fraction using appropriate detergents
- For Staphylococcus aureus check on these literature [PMID 15822900], [PMID 7093255]
- Do not boil the sample with loading buffer (If you find and doubt aggregations - multiple, non specific bands)
- Change your membrane extraction protocol, Including increasing the initial culture volume (If nothing else works)
- Try to raise antibody using an expressed whole protein (If peptide based antibody does not work)
- Try to raise monoclonal antibody (If polyclonal antibody does not work)