Dear RG users,
I have created a protein construct for E. coli-based expression system that contains uncleavable C-terminal Myc tag (protein-KLEQKLISEEDL; initial KL left from cloning). I have tried HRP conjugated anti-Myc from thermo fisher (R951-25), and I didn't see any signal in the gel. Am I using the right anti-body? Anyone who had similar problem?
Best,
T.
Dear Goran,
I am producing protein in E. coli BL21(DE3)-RIPL strain and I can see a band on SDS-PAGE with mass corresponding to my protein. I need to detect it with Mac anti-body for in vitro pull down assay using three proteins. One carries His-tag (and will be used to pull down others on NiNTA), one carries FLAG-tag, and one carries Myc-tag.
Dear Goran,
I am doing pull downs from clarified lysate. I load on the gel samples from input and elution. I prepared a large volume of input and elution sample and divided it into small fractions for SDS-PAGE. I transfer samples from three gels (one gel for His detection, one gel for FLAG detection, one gel for Myc detection). After transfers to a nitrocellulose, I can see bands for the other proteins (from gel for His and from gel for FLAG) but not Myc...
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