It's always better to use the secondary antibody specific to the first antibody used (if 1st Ab is mouse Ab - the secondary Ab should be anti-mouse IgG-HRP). If your secondary Ab is polyclonal and generated against whole IgG molecule then your rabbit Ab recognizing your 1st mouse Ab might be due to the recognition of the common Kappa Light Chain region 

Hi Viktor,

Thanks for the reply. Yes, i am using secondary antibody specific to the primary antibody. However, i will always observe the two bands when during x ray detection, but not during colorimetric detection.

I am using Santa Cruz secondary antibody HRP by the way. Do you think its better to change to another company's secondary antibody HRP? Or could it be due to the streptactin binding to somthing else which gives rise to the 2 bands?

Do you see the same bands when you omit the primary antibody?

Hi Adam,

I will let you know tomorrow as i am going to test by omitting the primary