Why is there still a heavy chain signal appearing on my blot?
When there is still an apparent heavy chain signal, this usually indicates that a complete reduction of the sample was not achieved before loading on the gel. A complete and sustaining reduction must be maintained for the ReliaBLOT system to effectively block detection of heavy and light chain signals. A complete and sustaining reduction is achieved by using 0.1M DTT in the sample buffer and loading the sample immediately after heating. Make sure your stock of DTT in solid form is being stored properly. If you are using frozen aliquots of DTT stock, do not thaw and refreeze.
Will the ReliaBlot kit work with mouse antibodies?
The ReliaBLOT® protocol is designed for use with primary (blotting) antibodies raised in rabbits. The protocol will not work with antibodies generated in other species.
Can I use an alternate HRP conjugate for the secondary antibody step?
No, the ReliaBLOT protocol will only work effectively when using Goat anti-Rabbit Light Chain HRP Conjugate (Bethyl, Cat. No, A120-113P).
Can I substitute another recipe for the blocking buffer/agent?
The ReliaBLOT protocol works most effectively with the Blocking Solution recommended.