PNGase F Kit #52749
Product Information
Product Usage Information
Denaturing Reaction Conditions:
1. Add 1-20 μg of glycoprotein, 1 μL of Glycoprotein Denaturing Buffer (10x), and H2O (if necessary) to make a total volume of 10 μL.
2. Heat reaction at 100°C for 10 min to denature glycoprotein.
3. Chill reaction on ice and centrifuge for 10 sec.
4. Add 2 μL of GlycoBuffer II (10x), 2 μL of 10% NP-40, and 6 μL of H2O to make a total reaction volume of 20 μL.
Note: PNGase F is inhibited by SDS, therefore it is essential to have NP-40 in the reaction mixture under denaturing conditions. Failure to include NP-40 in the denaturing protocol will result in loss of enzymatic activity.
5. Add 1 μL of PNGase F, mix gently.
6. Incubate at 37°C for 1 hr.
7. Analyze by method of choice.
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.
Note: The optimal incubation times and enzyme concentrations must be determined empirically for each individual substrate.
Non-Denaturing Reaction Conditions:
When deglycosylating a native glycoprotein, it is recommended that an aliquot of the glycoprotein is subjected to the denaturing protocol to provide a positive control for the fully deglycosylated protein. The non-denatured reaction can then be compared to the denatured reaction to determine the extent of reaction completion.
1. Add 1-20 μg of glycoprotein, 2 μL of Glycoprotein Denaturing Buffer (10x), and H2O (if necessary) to make a total volume of 20 μL.
2. Add 2-5 μL of PNGase F, mix gently.
3. Incubate at 37°C for 4-24 hr.
Note: To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
4. Analyze by method of choice.
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.
1. Add 1-20 μg of glycoprotein, 1 μL of Glycoprotein Denaturing Buffer (10x), and H2O (if necessary) to make a total volume of 10 μL.
2. Heat reaction at 100°C for 10 min to denature glycoprotein.
3. Chill reaction on ice and centrifuge for 10 sec.
4. Add 2 μL of GlycoBuffer II (10x), 2 μL of 10% NP-40, and 6 μL of H2O to make a total reaction volume of 20 μL.
Note: PNGase F is inhibited by SDS, therefore it is essential to have NP-40 in the reaction mixture under denaturing conditions. Failure to include NP-40 in the denaturing protocol will result in loss of enzymatic activity.
5. Add 1 μL of PNGase F, mix gently.
6. Incubate at 37°C for 1 hr.
7. Analyze by method of choice.
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.
Note: The optimal incubation times and enzyme concentrations must be determined empirically for each individual substrate.
Non-Denaturing Reaction Conditions:
When deglycosylating a native glycoprotein, it is recommended that an aliquot of the glycoprotein is subjected to the denaturing protocol to provide a positive control for the fully deglycosylated protein. The non-denatured reaction can then be compared to the denatured reaction to determine the extent of reaction completion.
1. Add 1-20 μg of glycoprotein, 2 μL of Glycoprotein Denaturing Buffer (10x), and H2O (if necessary) to make a total volume of 20 μL.
2. Add 2-5 μL of PNGase F, mix gently.
3. Incubate at 37°C for 4-24 hr.
Note: To deglycosylate a native glycoprotein, longer incubation time as well as more enzyme may be required.
4. Analyze by method of choice.
Note: The simplest method of assessing the extent of deglycosylation is by mobility shifts on SDS-PAGE gels.
Storage
All components in this kit are stable for 24 months when stored at the recommended temperature. Aliquot to avoid repeated freeze/thaw cycles.
Product Description
The PNGase F Kit provides reagents that, when combined, remove high mannose N-glycans from glycoproteins, leaving N-glycan core oligosaccharides intact and suitable for further analysis. Peptide N-Glycosidase F (PNGase F) is free of proteases and Endo F activities and is purified from Flavobacterium meningosepticum. It cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins. The PNGase F Kit can be used under native and denaturing conditions.
| Activity | Unit Definition: One unit is defined as the amount of enzyme required to remove > 95% of the carbohydrate from 10 µg of denatured RNase B in 1 hr at 37°C in a total reaction volume of 10 µL. |
Specificity / Sensitivity
The PNGaseF Kit effectively removes high mannose N-glycans from glycoproteins. It is non-recombinant with no detectable endoglycosidase F1, F2, or F3 contamination.
Source / Purification
The PNGase F component is produced in a strain of Flavobacterium meningosepticum and is free of proteases and Endo F activities.
限制使用
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For Research Use Only. Not For Use In Diagnostic Procedures.
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