Description
Product Usage Information
Denaturing Reaction Conditions: 1. Add 1-20 μg of glycoprotein, 1 μL of Glycoprotein Denaturing Buffer (10x), and H 2 O (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 H 2 O 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 H 2 O (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.
Specificity / Sensitivity
The PNGase F Kit effectively removes high mannose
Source / Purification
The PNGase F component is produced in a strain of and is free of proteases and Endo F activities.
