dye-free gel loading buffer ii  (Thermo Fisher)

Journal: Experimental & Molecular Medicine

Article Title: Proteins are a source of glycans found in preparations of glycoRNA

doi: 10.1038/s12276-025-01575-1

Figure Lengend Snippet: a A schematic representation of the glycoRNA labeling and purification strategy, following the procedure described by Flynn et al. . b A northern blot of RNA extracted from metabolically labeled 3T3 and HeLa cells. RNA was purified as outlined in a and conjugated to DBCO–biotin. Methylene blue was used to detect RNA on the nitrocellulose membrane. A streptavidin–peroxidase probe was used to detect the labeled glycans conjugated to DBCO–biotin. c The in-gel fluorescence detection of labeled glycans in preparations of RNA extracted from metabolically labeled 3T3 and HeLa cells. RNA was purified as in a and conjugated to DBCO–AF647. Following gel electrophoresis, the labeled glycans were visualized directly in the agarose gel. EtBr staining serves as a loading control. The biological triplicates are shown. d The RNase A/T1 treatment of RNA extracted from metabolically labeled HeLa cells. The first two lanes show undigested controls in ultrapure water or in digestion buffer. After the incubation with RNase A/T1, the samples were split. One half was left untreated, and the other was purified using silica columns. Three replicates of individual digestions are shown.

Article Snippet: Typically, 9 μl of RNA was mixed with 10 μl of dye free gel loading buffer (df-GLB; final concentration: 50% formamide, 9 mM ethylenediaminetetraacetic acid and 0.0125% SDS) and 1 μl of DBCO–PEG 4 –biotin (Jena Bioscience) or DBCO–AF647 (Jena Bioscience).

Techniques: Labeling, Purification, Northern Blot, Metabolic Labelling, Membrane, Fluorescence, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Staining, Control, Incubation

Journal: Experimental & Molecular Medicine

Article Title: Proteins are a source of glycans found in preparations of glycoRNA

doi: 10.1038/s12276-025-01575-1

Figure Lengend Snippet: a Western blot analysis of the purified sol-hLAMP1 contruct compared with the endogenous LAMP1 obtained from a HeLa cell lysate. b Western blot analysis of sol-hLAMP1 extracted via TRIzol or bound directly to a silica column. For the direct column loading, the samples were mixed with the silica column’s RNA binding buffer (Zymo) and additional volumes of ethanol or isopropanol as indicated. In the case of the TRIzol treatment, sol-hLAMP1 was extracted from the aqeuous phase and mixed with two volumes of isopropanol. All samples were then purified using silica columns according to the manufacturer’s protocol and analyzed by western blotting. A representative blot of two independent experiments ( n = 2) is shown. c A western blot analysis of sol-hLAMP1 obtained from silica column purifications in the presence of intact RNA or increased levels of isopropanol, analogous to Fig. . Sol-hLAMP1 was mixed with RNA and/or RNase A/T1 and incubated at 37 °C for 45 min. The aliquots were separated (‘Input’), and the rest was mixed with RNA binding buffer (Zymo) and isopropanol as indicated. The asterisk indicates the inactivation of RNase A/T1 by guanidine salts before adding RNA. The silica column purification was carried out following the manfacturer’s protocol. A representative blot of two independent experiments ( n = 2) is shown. d The agarose gel with EtBr staining of the samples shown in c .

Article Snippet: Typically, 9 μl of RNA was mixed with 10 μl of dye free gel loading buffer (df-GLB; final concentration: 50% formamide, 9 mM ethylenediaminetetraacetic acid and 0.0125% SDS) and 1 μl of DBCO–PEG 4 –biotin (Jena Bioscience) or DBCO–AF647 (Jena Bioscience).

Techniques: Western Blot, Purification, RNA Binding Assay, Incubation, Agarose Gel Electrophoresis, Staining