![]() Reduce the quantity of wasing steps or the time per step. The membrane was washed too excessively.If using PVDF, please keep in mind that the membrane needs to be activated with methanol prior to blotting. Make sure that the transfer was not performed the wrong way. Check the transfer with a reversible stain such as Ponceau S. The transfer of the protein to the membrane was poor.prepare nuclear lysates for a nuclear protein. Use an enrichment step to maximize the signal, e.g. The protein of interest is not abundantly present in the tissue.Use protease inhibitors during the preparation, and load at least an aggregate of 20-30 μg protein per lane. The amount of antigen could be insufficient.Please keep in mind that milk cannot be used for blocking if the detection should occur with Strep-Tactin® conjugates, since it contains biotin which is detected by Strep-Tactin®, and therefore produces high background. Use a mild detergent such as Tween 20 or switch blocking reagent. Cross-reaction between blocking agent and primary or secondary antibody.Use higher antibody concentrations or incubate longer, e.g. Not enough primary or secondary antibody is bound to the protein of interest.Alternatively, conjugated Strep-Tactin can be used for Strep-tagged proteins avoiding incompatible antibody pairs. primary is raised in rabbit, use anti-rabbit secondary). Use a secondary antibody that was raised against the species, in which the primary was raised (e.g. The primary antibody and the secondary antibody are not compatible.Please always include a positive and a negative control to check if the antibody or Strep-Tactin® conjugate is functional. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |