Hi All,
Pol II RPB1 CTD is phosphorylated, and can be detected as doublet bands on gel. I expressed a Myc tagged Rpb1 in cell, and tried to detect Rpb1-Myc. However, I can only see one band with anti-Myc antibody. Does anyone have an explanation for this?
sample preparation: I prepare the cell lysis by boiling cells in 1x sample buffer at 94 degree for 5 min. I ran the samples on a 6% gel.
Thank you for the help!
Hi there,
What is the detection used for the doublet? Anti-rpb1? If the heaviest band is positive with anti-myc detection it simply means the lower band has obviously lost the tag.
Dominique Liger In other studies, a doublet can be detected with both anti-CTD, or with an antibody against the tag on Rpb1.
I am not sure whether the band I observed is the higher mobility band or the lower mobility band (unphosphorylated vs phosphorylated Rpb1). Also, the tag is on the C-terminus.
Unless you detect the positive shift due to phosphorylation with an anti-phosphoSer antibody and compare it with the result obtained with anti-rpb1(or anti-CTD), you can't be sure of the nature of the single band you get with anti-myc detection alone (apart from it is containing myc epitope, is it full length? Is it phosphorylated/not phosphorylated? ). Mw estimate of the band should help you a little bit...
Dominique Liger Thank you for the suggestions! I actually treated the cell lysate with calf intestinal phosphatase. There is no difference between the treated and untreated samples. So I guess the single band I observed is the unphosphorylated form. It could also be a degradation product.
I am guessing during cell lysis, the endogenous phosphatase dephosphorylated Rpb1, so I could only detect a single band. I am trying a new lysis protocol.
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