Indicated cell lines (0.15 million cells/well) were plated on poly-lysine (0.15mg/mL) coated 24well plates and transiently transfected with 400ng indicated plasmids. Protein expression was induced with 200ng/mL doxycycline. After 24 h of transfection, the cells were collected in 150μL of 150ul 2x SDS sampled buffer and boiled for 10min. 20ul sample was mixed with 65ul of water, 5ul of 20% Triton X-100, 10 ul of 10X G5 buffer (NEB), and 1ul of Endo H HF (NEB). The reactions were incubated for 4h at and were terminated by adding 50μL 5x SDS sample buffer and boiled for 5 min before analyzing by immunoblotting. The above procedure was used for Endo H digestion of immunoprecipitated samples that were already boiled in a 2x SDS sample.
G5 buffer
Manufactured by New England Biolabs
Sourced in United Kingdom
The 10X G5 buffer is a concentrated solution designed for use in various DNA and RNA manipulation experiments. It provides the necessary ionic conditions and pH environment to support the optimal performance of numerous enzymatic reactions, including DNA amplification, restriction digestion, and ligation. The 10X G5 buffer is a versatile solution that can be diluted to the desired working concentration prior to use.
Automatically generated - may contain errors
Lab products found in correlation
Endoglycosidase h, by Roche (2 mentions)
Concentrated protease inhibitor cocktail, by Roche (2 mentions)
Endo h hf, by New England Biolabs (1 mentions)
G7 buffer, by New England Biolabs (1 mentions)
Hybond c, by GE Healthcare (1 mentions)
Endo h, by New England Biolabs (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
Hybond, by Cytiva (1 mentions)
4 protocols using g5 buffer
1
Endo H Digestion of Immunoprecipitated Proteins
2
Endoglycosidase Digestion of BY2 Cell Extracts
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Endoglycosidase digestion was performed according to 37 with a few modifications. Cell extracts from BY2 lines transformed with pTRA-CardB were prepared as previously described. Then, 3 L of 10X concentrated denaturing buffer (500 mM sodium citrate, pH 5.5, 2% SDS, 10% mercaptoethanol) was added to 30 L of cell extract and samples were boiled at 100 °C for 10 min. After boiling, samples were incubated for 5 min on ice and 3 L of 10X G5 buffer (New England BioLabs, UK) were added to the samples together with 1.5 L of 25X concentrated protease inhibitor cocktail (Roche, USA) and endoglycosidase H (2.5 mU) (Roche, USA). Samples were incubated at 37 °C overnight. Control samples were treated without endoglycosidase H. Finally, 0.25 volumes of 4X SDS-PAGE loading buffer was added to the samples and analyzed by SDS-PAGE and western blot as previously described.
3
Deglycosylation of Immunoglobulin Proteins
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SDS-PAGE was performed in 8–10% polyacrylamide gels run under reducing or non-reducing conditions. Separated proteins were either detected by Coomassie Brilliant Blue staining or by transfer onto nitrocellulose membranes (Hybond-C, GE Healthcare) and subsequent detection with different antibodies and chemiluminescence-based detection reagents. Detection of the αC was done using a polyclonal goat anti-human alpha chain specific antibody (Sigma–Aldrich), the λC was detected using a rabbit anti-human lambda light chain antibody (Sigma–Aldrich) and the SC was detected using a rabbit anti-human SC antibody (Gentaur).
Crude protein extracts, SSL7-prufied sIgA1 or IF fractions were subjected to enzymatic deglycosylation. For endoglycosidase H (Endo H) digestion 1.5 μl of 10x Glycoprotein Denaturing Buffer (NEB, 5% SDS, 0.4 M DTT) were added to 13.5 μl of sample. This mix was incubated for 10 min at 95°C. After the sample had cooled down on ice, 2 μl G5 Buffer (NEB), 1 μl Endo H (NEB) and 2 μl ultrapure water were added and this mix was incubated for 60 min at 37°C. For the peptide: N-glycosidase F (PNGase F) digestion 1.5 μl of denaturing buffer were added to 13.5 μl of sample. This mix was incubated for 10 min at 95°C. After the sample had cooled down on ice, 2 μl G7 Buffer (NEB), 1 μl PNGase F (NEB), and 2 μl NP-40 were added and this mix was incubated for 60 min at 37°C.
Crude protein extracts, SSL7-prufied sIgA1 or IF fractions were subjected to enzymatic deglycosylation. For endoglycosidase H (Endo H) digestion 1.5 μl of 10x Glycoprotein Denaturing Buffer (NEB, 5% SDS, 0.4 M DTT) were added to 13.5 μl of sample. This mix was incubated for 10 min at 95°C. After the sample had cooled down on ice, 2 μl G5 Buffer (NEB), 1 μl Endo H (NEB) and 2 μl ultrapure water were added and this mix was incubated for 60 min at 37°C. For the peptide: N-glycosidase F (PNGase F) digestion 1.5 μl of denaturing buffer were added to 13.5 μl of sample. This mix was incubated for 10 min at 95°C. After the sample had cooled down on ice, 2 μl G7 Buffer (NEB), 1 μl PNGase F (NEB), and 2 μl NP-40 were added and this mix was incubated for 60 min at 37°C.
4
Endoglycosidase Digestion of BY2 Extracts
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Endoglycosidase digestion was performed as previously described35 (link) with a few modifications. Cell extracts from BY2 lines transformed with pTRA-CardB were prepared as previously described. Then, 3 μL of 10X concentrated denaturing buffer (500 mM sodium citrate, pH 5.5, 2% SDS, 10% β-mercaptoethanol) was added to 30 μL of cell extract and samples were boiled at 100 °C for 10 min. After boiling, samples were incubated for 5 min on ice and 3 μL of 10X G5 buffer (New England BioLabs, UK) were added to the samples together with 1.5 μL of 25X concentrated protease inhibitor cocktail (Roche, USA) and endoglycosidase H (2.5 mU) (Roche, USA). Samples were incubated at 37 °C overnight. Control samples were treated without endoglycosidase H. Finally, 0.25 volumes of 4X SDS-PAGE loading buffer were added to the samples and analyzed by SDS-PAGE and western blot as previously described.
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