Revision 1

#52749

Store at -20C

CST Logo
Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

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.

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 N-glycans from glycoproteins.This kit 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.

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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

Limited Uses

Except as otherwise expressly agreed in a writing signed by a legally authorized representative of CST, the following terms apply to Products provided by CST, its affiliates or its distributors. Any Customer's terms and conditions that are in addition to, or different from, those contained herein, unless separately accepted in writing by a legally authorized representative of CST, are rejected and are of no force or effect.

Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 1

Cell Signaling Technology Logo
Western blot analysis of extracts from mouse bone marrow-derived macrophages (BMDM), RAW 264.7, and CRISPR/Cas9 TREM2 knockout (KO) RAW 264.7 cells, untreated (-) or treated with Peptide N-Glycosidase F (PNGase F; +), using TREM2 (E9O9F) Rabbit mAb #59621 (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
undefined Image 1: PNGase F Kit
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.