A dot blot will provide a qualitative estimate of antibody titer, as described in this protocol. The same test can be done quantitatively by enzyme-linked immunosorbent assay (ELISA), but the technically simpler dot blot gives adequate information, uses less protein, and does not require an ...
We have developed a dot-blot assay specifically for use with a LiCor Odyssey CLx imager, which allows for sensitive detection of proteins in the infrared range. Here, we provide a detailed protocol for the preparation of brain tissue and neural cell culture lysates for analysis of protein ...
of large proteins. Transfer efficiency can be checked by staining proteins on the membrane using Ponceau S (Protocol 8, page 79). Once transferred to the membrane, the proteins can be probed with epitope-specific antibodies or conjugates.For buffer and reagent compositions, see “Western Transfer ...
1d), and the comparable performance on conversional dot blot assay (Supplementary Fig. S5–6). By using the TotalSeq-A antibody on protein samples under the established protocol, we showed that the TotalSeq assay gives comparable data with current protein quantification methods, such as western ...
There could not have been any dilution of samples in the collection protocol, but surface evaporation in the cage could have imposed variable concentration. Table 1 Urinary testosterone of Wistar male rats expressed adjusted according to associated creatinine concentration Full size table Table 2 ...
2a–c). Omission of any component or step in the PDPL protocol resulted in significant reversion of signal-to-background (Fig. 1d). Notably, protein labeling was greatly reduced in the presence of sodium azide or trolox, which are known to quench singlet oxygen28. The presence of D2O, ...
Proteins were transferred onto nitrocellulose membranes using the iBlot system (Life Technologies), with a standard (P0, 8 min) protocol. Nitrocellulose was blocked for 60 min in 5% w/w non-fat dried milk (Marvel) in Tris buffered saline/0.05% Tween-20 (TBST). Primary antibodies used ...
(1 × 106 cells/ well) treated with vehicle (control (C), n = 3) or oligopyridylamides (5 µM ADH-1 or ADH-6, n = 3) using a combination of both TriZol (Thermo Fisher Scientific) and RNAeasy Mini Kit (Qiagen) with modification of the manufacturers’ protocol. ...
Using a modified version of a previously established protocol11 (Extended Data Fig. 1), we assembled the GABAB–Gil complex by incubating purified GABAB with Gi1 in the presence of the agonist baclofen and the PAM R,S-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (BHF...
meningitidis ProQ, we adopted an in vivo UV CLIP-seq protocol3,4 for analysis of cross-linked RNA-protein complexes after UV light irradiation of live bacteria (Supplementary Figs. 3, 4a). Meningococci expressing C-terminal 3× FLAG tag ProQ from the native locus were grown in rich medium...