User:Vijay/Trouble Shooting/western

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Just Suggestions

  1. Reduce protein load in the gel (If you get too many/unwanted banding, in addition to your band)
  2. Increase protein load in the gel (If you do not see your band)
  3. Use higher percentage gel
  4. Wash properly (If too much of background)
  5. Check on the blocking step (If too much of background)
  6. Reduce or increase the concentration of the primary/secondary antibody (If you get excess or no signal for your band)
  7. Reduce or increase the exposure time after developing the film (If you get poor/strong signal)
  8. 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...

  1. Solubilize the membrane fraction using appropriate detergents
  2. For Staphylococcus aureus check on these literature [PMID 15822900], [PMID 7093255]
  3. Do not boil the sample with loading buffer (If you find and doubt aggregations - multiple, non specific bands)
  4. Change your membrane extraction protocol, Including increasing the initial culture volume (If nothing else works)
  5. Try to raise antibody using an expressed whole protein (If peptide based antibody does not work)
  6. Try to raise monoclonal antibody (If polyclonal antibody does not work)