We are currently trying to work out conditions for using your anti-ICP4 antibody (H1A021-100). We infected Vero cells with wild type HSV for 18hrs scraped, pelleted and boiled the pellet in SDS prior to loading on a 10% SDS-PAGE. The PVDF membrane was immunostained with ICP4 Ab overnight 4oC in 5% milk and .05% azide at 1:500. When the blot was detected there was a slight band around 175kD but the whole lane was dark with some bands. There was no background staining in the lanes that had uninfected Vero cells or Vero infected with mutant HSV(no ICP4). Do you have any suggestions as to why I am not seeing a nice conclusive band where I should (@175kD)?
Resolution:
My guess is that you need to reduce some concentrations somewhere....
We have had similar issues in the past in using other antibodies - that is one of the reasons we are so careful to know the amount of antigen we apply to the PVDF. Our standard is 10 µg per cm lane width.
My suggestion would be to reduce the antigen load - perhaps several variations simultaneously. If you are using chemiluminescence, I might reduce the antibody concentration too.
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