Hi there,
After a good previous experience using the O-GlcNAc-Specific Antibody CTD110.6, WBs do not work anymore. In particular a negative control (previous used) and a new negative control (recently added to our experiments) are both recognized by this antibody.
Here goes my conditions and a WB picture of the problem, in case someone can help.
Thanks in advance ,-)
- Nitrocellulose membrane (Pall) 0.2 µm.
- Blocking 1h RT in TBS 1X + Tween 20 (0.1 %) + BSA (Bovine Serum Albumin Fraction V, Merck) 10 %.
- Primary Ab --> Anti-O-GlcNAc CTD110.6 (Sigma) (1/2000) in TBS 1X + Tween 20 (0.1 %) + BSA 10 %. Incubated ON (4 ºC).
- Wash 3 times with TBS 1X + Tween 20 (0.1%).
- Secondary Ab --> HPR conjugate IgG Sheep anti-Mouse (Amersham), diluted 1:10 000 in TBS 1X + Tween 20 (0.1 %) + BSA 10 %. Incubate 2h (R.T.) Same, or even worst, results are obtained using a more specific secondary antibody (m-IgGκ BP-HRP (sc-516102) diluted 1/2500, incubated 1h at RT).
- Wash 3 times with TBS 1X + Tween 20 (0.1%).
- Chemiluminescent development by Clarity ECL Western Blotting Substrate.
Two types of negative controls have been included.
(1) Plant virions unable to O-GlcNAcylation
(2) Total protein from yeast (S. cereviceae), OGT lacking organisms. Specially for yeast a null O-GlcNAcylation signal has been reported in several publications.
In both cases, no signal is expected. However, WB shows an CTD110.6 over-recognition and negative control failures.
https://www.frontiersin.org/articles/10.3389/fragi.2020.620382/full
This review article may help you.
In addition to labeling O-GlcNAc, CTD110.6 also recognizes N-GlcNAc2-modified proteins (Isono, 2011) and terminal β-glycans on the complex N-glycans.
Thank you very much Chandru, I noted your recommendation. I'm afraid the background trouble is a general problem. Thanks again.
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