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Glycoprotein Detection with the Odyssey Infrared Imaging System

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Glycoprotein Detection with the Odyssey Infrared Imaging System Glycoprotein Detection with the Odyssey ® Infrared Imaging System Julie A. Champoux, Kristi L.H. Ambroz, Joseph B. Hwang, William M. Volcheck, and Amy R. Schutz-Geshwender LI-COR Biosciences, Lincoln, NE 68504 INTRODUCTION bination of lectins can also be used to dissect the Glycosylation is one of the most common and carbohydrate composition of a target protein. important events in post-translational modifica- For more information about IRDye products tion, with over half of all proteins believed to be used in this study, go to http://www.licor.com/bio/ glycosylated.1 Cellular glycoconjugates play reagents/irdyes.jsp. important roles in many biological processes and have been implicated in cancer development, MATERIALS AND METHODS retrovirus infection and other diseases. Carbohy- Sensitivity of biotinylated Con A / IRDye drate-binding proteins known as lectins bind to 800CW-streptavidin and IRDye 800CW-Con A specific oligosaccharides, and can serve as markers Two-fold serial dilutions of α2-macroglobulin, to identify certain cell types or cellular compo- glucose oxidase and RNAse B (Sigma Cat# nents. Lectins have very high binding specificity M6159, 49178 and NEB Cat# P7817S were and have been used to characterize and purify mixed and separated on 4-12% polyacrylamide oligosaccharides.2 This application note describes Tris-glycine gels (Novex/Invitrogen). Samples the use of lectins to detect glycoproteins in West- were electrophoretically transferred onto nitro- ern blot format using the Odyssey® Infrared cellulose membrane (Osmonics). For all blots in Imaging System. this study, blocking was carried out with Odyssey Concanavalin A (Con A) is a carbohydrate- Blocking Buffer (LI-COR, Part # 927-40000) for 1 h binding protein that binds specifically to the at room temperature; washing was performed in most commonly occurring sugars: α-D-mannose, PBST (PBS + 0.1% Tween®-20) for 4 x 5 min, fol- α-D-glucose and, with lower affinity, α-N-acetyl- lowed by a 5 min rinse in PBS before imaging. glucosamines (GlcNAcs)3,4. Commercially avail- One blot was incubated with biotinylated Con A able biotinylated Con A and IRDye® 800CW- (Vector Laboratories, Cat# B1005) diluted 1:1000 streptavidin (LI-COR, Part # 926-32230) were in Odyssey Blocking Buffer + 0.2% Tween-20 for used to detect various glycoproteins by Western 3 h. The blot was washed, then incubated in a analysis. Infrared detection with biotin/IRDye- 1:10,000 dilution of IRDye 800CW-streptavidin streptavidin and the Odyssey system is robust (LI-COR, Part # 926-32230) for 1 h, then washed and sensitive with a detection limit of 4-8 ng, and again. The blot was imaged with the Odyssey eliminates the need for colorimetric and chemilu- Infrared Imaging System. minescent reagents. Con A can also be covalently As an alternative to biotin-streptavidin detec- labeled with IRDye® 800CW (using one of tion, Con A (concanavalin A; EY Laboratories, LI-COR's IRDye® 800CW Protein Labeling Kits, Inc., Cat. # L1104) was directly conjugated with Part # 928-38040 or 928-38044) and used to amine-reactive IRDye 800CW (IRDye 800CW Pro- detect glycoproteins directly, with a detection limit tein Labeling Kit Part # 928-38040) and purified of 8-16 ng on a Western blot. By adding an anti- by dialysis. A dye/protein ratio of 1.7 was body against the protein of interest in combination obtained. The resulting IRDye 800CW-Con A with a lectin, two-color detection can be performed (1 mg/ml) was diluted 1:1000 and used to detect on cell lysates to detect the target protein and an identical blot. The blot was washed, then make inferences about glycosylation state. A com- imaged with Odyssey. Published January, 2006 by LI-COR Biosciences www.licor.com 1 Champoux, et al. Detection of purified glycoproteins using with 1:1000 mouse anti-human EGFR as above. biotinylated Con A/IRDye 800CW-streptavidin After washing, they were incubated with 1:2500 Several purified proteins were digested with Pep- Alexa Fluor® 680 goat-anti-mouse and 1:1000 tide: N-Glycosidase F (PNGase F; New England IRDye 800CW-Con A, IRDye 800CW-WGA, or BioLabs, Cat. # P0704S) which cleaves between IRDye 800CW-UEA I (all 1 mg/ml). After wash- the innermost GlcNAc and asparagine residues ing, blots were imaged with Odyssey in the 700 of N-linked glycoproteins. Undigested and nm (total EGFR) and 800 nm (lectin) channels. digested proteins (20 µg each) were separated on 4-12% gels. One replicate gel was stained with RESULTS AND DISCUSSION Coomassie™ (Invitrogen SimplyBlue™ Protein Biotinylated Con A is a convenient reagent that Stain) to detect total protein; another was trans- facilitates straightforward detection of glycopro- ferred to nitrocellulose. After blocking, blots were teins using IRDye 800CW-streptavidin to visual- incubated for 1.5 h with 1:1000 biotinylated Con ize Con A binding. This technique was used to A in Odyssey Blocking Bufffer + 0.2% Tween-20, detect several purified glycoproteins (Fig. 1A). washed, incubated with 1:10,000 IRDye 800CW- As an alternative, Con A was conjugated with streptavidin for 1 h, and washed again. Blots and IRDye 800CW by a standard amine-directed gels were imaged with Odyssey, using the 800 labeling chemistry, generating a directly labeled nm channel to image IRDye 800CW and the 700 lectin reagent that streamlines the detection nm channel to image Coomassie staining. process (Fig. 1B). Sensitivity of both detection methods was determined in Western blot format Detection of EGFR in A431 cell lysates using using three known glycoproteins: α2-macroglobu- human anti-EGFR / AlexaFluor® 680 anti- lin from human plasma5 (180 kDa), glucose oxi- body and IRDye 800CW-Con A dase from A. niger6 (82 kDa), and RNAse B from Undigested and PNGase F-digested A431 cell bovine plasma7 (17 kDa). The limits of detection lysates (25 µg each) were separated on replicate with biotinylated Con A/IRDye 800CW-streptavi- 4-12% gels. One gel was stained with Coomassie din and IRDye 800CW-Con A were 4-8 ng and 8-16 to confirm equal sample loading, and the other ng, respectively. was transferred to nitrocellulose. After blocking, In Fig. 2A, a selection of purified proteins blots were incubated with 1:1000 mouse anti- were examined in greater detail. Protein sam- human EGFR (Biosource, Cat. # AHR5062) in ples were treated with PNGase F, a glycosidase Odyssey Blocking Buffer + 0.2% Tween-20 over- which removes all terminal and internal night at 4 °C. Blots were washed, then incubated N-linked glycosylation. A shift in migration of simultaneously with 1:2500 Alexa Fluor® 680 the band in digested samples indicates that goat-anti-mouse (Molecular Probes Cat. # A21057) N-linked oligosaccharides were present in the and 1:1000 IRDye 800CW-Con A for 1 h. After sample. Band shifts were observed for α2-macro- washing, blots were imaged with Odyssey in the globulin, glucose oxidase, and transferrin. Phos- 700 nm (total EGFR) and 800 nm (Con A) phorylase and BSA did not show a band shift, channels. implying a lack of N-linked glycosylation. Data Characterization of EGFR in normal and were consistent with published reports5, 6, 8, 9, 10. cancer cell lysates using labeled lectins In Fig. 2B, proteins were transferred to nitro- The lectins Con A, WGA (wheat germ agglutinin; cellulose for Western analysis and probed with EY Laboratories, Inc., Cat. # L2101) and UEA-I biotinylated Con A. After detection with IRDye (Ulex europaeus; EY Laboratories, Inc., Cat. # 800CW-streptavidin, α2-macroglobulin and glu- L-2201) were directly conjugated with IRDye cose oxidase showed strong bands in the undi- 800CW using standard amine-directed labeling gested samples and the absence of corresponding techniques, and purified by dialysis. bands in the digested samples, indicating that Normal epidermal and cancer epidermal these proteins contain carbohydrate residues (A431) lysates were separated on 4-12% gels, capable of binding Con A. Phosphorylase and then stained with Coomassie or transferred to BSA are not glycoproteins and were not detected nitrocellulose. Blots were blocked, then incubated by Con A. Interestingly, although a band shift 2 LI-COR Biosciences Glycoprotein Detection with the Odyssey Infrared Imaging System A B Fig. 1. Western blot of two-fold serial dilutions of α2-macroglobulin, glucose oxidase and RNAse B from 2 µg to 244 pg. Limit of detection for glucose oxidase was 4 ng (lane 10) with biotinylated Con A / IRDye 800CW-strepta- vidin (panel A), and 8 ng (lane 9) with IRDye 800CW-Con A (panel B). 2-macroglobulin α2-macroglobulin α phosphorylaseglucose oxidaseBSA transferrin phosphorylaseglucose oxidaseBSA transferrin PNGaseF - + - + - + - + - + - + - + - + - + - + AB Fig. 2. A) Coomassie stain of untreated and PNGase F-treated proteins. B) Western blot showing undigested (-) and PNGase F-digested (+) proteins. Blot was probed with biotinylated Con A and detected with IRDye 800CW- streptavidin. was observed for transferrin after digestion with tant to note that some lectins exhibit non-specific PNGase F, digested transferrin was still detected hydrophobic binding to nonglycosylated mole- by Con A. It is possible that the transferrin oli- cules. As shown here, specificity should always be gossacharide detected by Con A is coupled via confirmed using appropriate glycosidases.2 O-linkage and was therefore not digested by It would be very useful if this technique could PNGase F. Transferrin is predicted to have an not only yield information about glycosylation O-linkage in addition to N-linkage8. It is impor- state, but also confirm the identity of the glyco- www.licor.com 3 Champoux, et al. protein in a whole cell lysate using a specific skin tissue. The EGFR glycoprotein was detected antibody. To this end, epidermal growth factor as in Fig. 3, and oligosaccharide composition was receptor (EGFR), another glycoprotein11, was simultaneously characterized using different analyzed with a two-color detection methodology IRDye 800CW-conjugated lectins.
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