User:Vijay/Trouble Shooting/western: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
| Line 8: | Line 8: | ||
#Check on the blocking step (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 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) | #Try peptide blocking, if using a cocktail peptide (to minimize cross reactivity of the antibodies) | ||
Revision as of 13:53, 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 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]
- Change your membrane extraction protocol (including increasing the initial culture volume)
