Improved immobilization and conjugation of glycoproteins
Efficient aldehyde-hydrazide covalent coupling using a catalyst and an optimized buffer.
Scott Meier, M.S.;
Ryan Bomgarden, Ph.D.;
Kay Opperman, Ph.D.;
Chris Etienne, Ph.D.;
Barbara Kaboord, Ph.D.;
Aldehyde-hydrazide chemistry has been used for over 30 years for coupling oxidized glycoproteins to activated agarose supports. This chemistry is pH- and time-sensitive, and can result in low yields even after 48 hours of incubation. We have optimized the buffer conditions and added a catalyst to reduce the reaction time while maximizing coupling.
The Thermo Scientific GlycoLink Immobilization and IP Kits provide an efficient and robust method for coupling oxidized glycoproteins to a beaded resin. The method uses sodium meta-periodate to gently oxidize glycoproteins and convert sugar cis-diols to aldehydes. The aldehydes react with hydrazide groups on the beaded resin to form stable hydrazone bonds. Aniline is added to catalyze the reaction, resulting in greater than 90% coupling in 4 hours or less, depending on the amount of protein glycosylation. Antibodies containing sufficient glycosylation in the Fc region can be immobilized with optimal orientation and an unobstructed binding domain. The immobilized glycoprotein or antibody can be used for immunoaffinity purifications or studying a variety of protein-protein interactions.
The Thermo Scientific GlycoLink Coupling Catalyst is a buffered solution that contains aniline and is included with the immobilization and IP kits. It can also be purchased separately for performing carbonyl-reactive labeling reactions using alkoxyamine-biotin and hydrazide-biotin reagents.
We evaluated the coupling efficiency of several glycoproteins using the GlycoLink Immobilization Kits, as well as nonspecific binding of the resin included in the kits. We also evaluated the ability to immunoprecipitate several antigens and to isolate a protein-protein complex using the GlycoLink IP Kit. Finally, we evaluated the improvements of aldehyde-alkoxyamine coupling chemistry when using the GlycoLink Coupling Catalyst.
RESULTS and DISCUSSION:
Improved coupling is based on optimized pH conditions and aniline, which acts as a catalyst. These optimizations enable immobilization of a wide variety of glycoproteins, including molecules with low amounts of glycosylation (Table 1). The Thermo Scientific UltraLink Hydrazide Resin included in the GlycoLink Immobilization and IP Kits is an activated beaded-polyacrylamide support with higher binding capacity per milliliter and lower nonspecific binding compared to hydrazide agarose supports (Figure 1).
Table 1. Glycosylation properties of polyclonal antibodies (and ovalbumin) and efficiency of glycoprotein coupling to Thermo Scientific GlycoLink Resin. For each experiment, 0.4mg of protein was reacted with 0.1mL of resin.
||# Glycosylation Sites
||% Occupancy of Sites
|Monoclonal Rat IgG1
|Monoclonal Mouse IgG1
|Rabbit Serum IgG
|Polyclonal Chicken IgY
|Human Serum IgG
|Human Serum IgM
Figure 1. Comparison of hydrazide-activated supports. Panel A: UltraLink Hydrazide Resin, included in the GlycoLink Immobilization and IP Kits, binds more rabbit IgG per milliliter of resin than the hydrazide-activated agarose resin. Panel B: The UltraLink Hydrazide Resin results in less nonspecific background compared to hydrazide-activated agarose. Samples were separated by SDS-PAGE and silver-stained. MW: Molecular-weight marker.
To determine the specificity of an antibody coupled to UltraLink Hydrazide Resin, mouse IgG1 anti-HA antibody immobilized using the GlycoLink Micro Immobilization Kit was incubated with a bacterial lysate expressing HA-GST-GFP (50μg). A single band corresponding to the correct molecular weight of HA-GST-GFP (55kDa) was purified with minimal background (Figure 2; Panel A). To test for recovery, 50μg of chicken IgY anti-rabbit immobilized using the GlycoLink Micro Immobilization Kit was incubated with 10μg of purified rabbit IgG. Recovery of antigen was high, with no rabbit IgG detected in the flow-through (Figure 2; Panel B).
Figure 2. The Thermo Scientific UltraLink Hydrazide Resin enables high specificity and recovery of target antigen. Panel A: Immobilized mouse IgG1 anti-HA antibody was used to purify HA-GST-GFP from a bacterial lysate. Samples were separated by SDS-PAGE and stained. Lane 1: Bacterial lysate (2µL; 1mg/mL); Lane 2: Flow-through (15µL): Lanes 3-4: Wash (15µL); Lane 5: Elution (2µL). Panel B: Immobilized chicken anti-rabbit antibody was used to isolate rabbit IgG. Samples were separated by SDS-PAGE and silver-stained. Lane 1: Rabbit IgG (10μL; 1mg/mL); Lane 2: Flow-through (10µL); Lane 3: Wash (10µL); Lane 4: Elution (10µL). MW: Molecular-weight marker.
Using the GlycoLink IP Kit, several target antigens were immunoprecipitated. EGFR, a membrane-bound growth factor receptor, and STAT1, a transcription factor located in the the nucleus, were precipitated from 0.5mg of A549 lysate with the appropriate antibody coupled to 20µL of UltraLink Hydrazide Resin (Figure 3; Panel A). The GlycoLink IP Kit also effectively isolated the protein-protein complex of Cdk1 and cyclin B1 from 0.75mg of late G2-synchronized U2OS cell lysate (Figure 3; Panel B).
Figure 3. Western blot of immunoprecipitated and co-immunoprecipitated proteins. Panel A: STAT1 and EGFR immunoprecipitation using 2μg and 10μg of antibody immobilized onto 20µL of UltraLink Hydrazide Resin. Panel B: Co-immunoprecipitation of Cdk1-cyclin B1 complex using 10μg of anti-Cdk1 antibody immobilized onto 20µL of UltraLink Hydrazide Resin.
The GlycoLink Coupling Catalyst increases the formation of a hydrazone bond between oxidized glycoprotein and alkoxyamine-biotin. Sialic acid containing glycoproteins on the cell surface were selectively oxidized with 1mM sodium meta-periodate. Adding aniline to the coupling buffer increased labeling efficiency of sialylated glycoproteins with alkoxyamine-biotin by almost 50% (Figure 4). Adding aniline to the coupling buffer also increased efficiency of immobilization to the UltraLink Hydrazide Resin (Figure 5).
Figure 4. The Thermo Scientific GlycoLink Coupling Catalyst improves aldehyde-hydrazide/alkoxyamine coupling. CHO cells were treated with sodium meta-periodate (Part No. 20504) to selectively oxidize sialic acids. Oxidized cells were incubated with Alkoxyamine-PEG4-Biotin (Part No. 26137) in either coupling buffer or in the GlycoLink Coupling Catalyst (aniline). Biotinylated protein was detected by Western blotting and evaluated by densitometry.
Figure 5. Aniline increased binding efficiency of human IgG to hydrazide resin. Human IgG (10mg) was oxidized, diluted to 5mg/mL either with GlycoLink Coupling Buffer alone or with GlycoLink Coupling Catalyst and coupled to 1mL of hydrazide-activated UltraLink Hydrazide Resin. Results are the average of three separate immobilizations for each condition after incubating for 4 hours.
The coupling of glycoproteins to a bead support using the GlycoLink Immobilization Kits is highly effective for immunoaffinity purification of proteins, allowing for the coupling of any antibody isotype provided it is sufficiently glycosylated. Target antigens immunoprecipitated with the GlycoLink IP Kit can be used for downstream applications and for isolating protein-protein interactions in vivo by co-immunoprecipitation. GlycoLink Immobilization and IP Kits in conjunction with the GlycoLink Coupling Catalyst, provide optimal coupling and labeling of oxidized glycoproteins, resulting in a fast and efficient reaction.
Comparison of hydrazide-activated supports
Rabbit IgG (10mg) was coupled using the GlycoLink Immobilization Kit or to hydrazide agarose resin. Immobilized protein was quantified using Tech Tip #9: Quantitate Immobilized Protein. To evaluate nonspecific binding, 20μL of UltraLink Hydrazide Resin or hydrazide agarose resin was incubated with 0.4mg of HeLa lysate for 1 hour, washed three times with TBS-Tween-20* (Part No. 28360) and eluted with 100μL of IgG Elution Buffer (Part No. 21004). Eluted fractions (10μL) were separated by SDS-PAGE and stained with Thermo Scientific Pierce Silver Stain (Part No. 24612).
Purification of HA-GST-GFP and purified rabbit IgG
Mouse IgG1 anti-HA antibody (50μg) was immobilized using the GlycoLink Micro Immobilization Kit. Bacterial lysate (50μg) containing HA-GST-GFP fusion protein was diluted with 0.45mL PBS and incubated with coupled resin for 15 minutes at room temperature. The resin was washed two times with TBS-Tween-20, and the bound protein was eluted with 100μL of 3M sodium thiocyanate. Each fraction was separated by non-reducing SDS-PAGE and stained with Thermo Scientific Imperial Protein Stain (Part No. 24615).
Chicken anti-rabbit IgG (50μg) was immobilized using the GlycoLink Micro Immobilization Kit. Purified rabbit IgG (10μg) was diluted with 100μL of PBS and incubated with coupled resin for 30 minutes at room temperature. The resin was washed two times with TBS-Tween-20, and the bound protein was eluted with 100μL of 3M sodium thiocyanate. Each fraction (10µL) was separated by non-reducing SDS-PAGE and stained with Pierce Silver Stain (Part No. 24612).
Immunoprecipitation of STAT1 and EGFR
Polyclonal rabbit IgG anti-EGFR (Ab No. PA1-1110) or anti-STAT1 antibody (EMD Millipore) was coupled to 20μL of UltraLink Hydrazide Resin at a final amount of 2μg and 10μg, using the GlycoLink IP Kit. A549 cell lysate (0.5mg) was incubated with the coupled resin for 1 hour at room temperature. The flow-through was collected by centrifugation and the resin was washed three times with IP Lysis/Wash Buffer. Bound protein was eluted with 100μL of Elution Buffer. Load and elution fractions (10% final volume, each fraction) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Membranes were blocked and blotted with the appropriate antibody at 1μg/mL for 1 hour, and detected with goat anti-rabbit HRP antibody (Part No. 31460) and Thermo Scientific SuperSignal West Dura Substrate (Part No. 34076). Blots were imaged with a CCD camera and 10 second exposures.
Co-immunoprecipitation of cyclin B1-Cdk1 complex
Rabbit polyclonal anti-Cdk1 (10μg) was immobilized using the GlycoLink IP Kit. U2OS cells at 50% confluence were serum-starved for 48 hours and stimulated with media plus 20% fetal bovine serum for 16 hours. Cells were lysed using the IP Lysis/Wash Buffer with the Thermo Scientific Halt Protease and Phosphatase Inhibitor Cocktail (Part No. 78440) added. Cell lysate (0.75mg) was incubated with 20μL blank resin or 20μL anti-Cdk1-coupled resin overnight in a cold room. Anti-Cdk1-coupled resin (20μL) was also incubated with IP Lysis/Wash Buffer alone. After overnight incubation, the nonbound fraction was collected by centrifugation and the resin was washed three times with IP Lysis/Wash Buffer. Bound protein was eluted with 50μL of Lane Marker Non-reducing Sample Buffer (Part No. 39001). Fractions (10μL) were separated by SDS-PAGE, transferred to nitrocellulose membranes and blocked. The membranes were probed with mouse IgG1 anti-cyclin B1 or mouse IgG1 anti-Cdk1, and detected with goat anti-mouse HRP (Part No. 31430) and SuperSignal West Femto Maximum Sensitivity Substrate (Part No. 34095). Blots were imaged with X-ray film and 2 minute exposures.
Labeling and detection of sialylated glycoproteins
CHO cells transfected with a vector expressing beta-2 adrenergic receptor were grown to confluence in a 10cm tissue culture dish. Cells were washed three times with 2mL of ice-cold GlycoLink Coupling Buffer. The cells were treated with 2mL of 1mM sodium meta-periodate (Part No. 20504) in coupling buffer on ice for 30 minutes in the dark to selectively oxidize sialic acids. The cells were washed three times with 2mL of coupling buffer. To label the sialylated glycoproteins on the cell surface, the oxidized cells were incubated with 2mL of 100μM Alkoxyamine-PEG4-Biotin (Part No. 26137) in either coupling buffer or in GlycoLink Coupling Catalyst and incubated for 1 hour at room temperature. The cells were washed three times with 2mL of ice-cold PBS and lysed with 0.5mL of IP/Lysis Buffer with Halt Protease and Phosphatase Inhibitor. The lysates were quantitated with the Thermo Scientific Pierce BCA Protein Assay (Part No. 23225), separated (1μg) by SDS-PAGE, transferred to a nitrocellulose membrane and blocked. Biotinylated protein was detected using High Sensitivity Streptavidin-HRP (Part No. 21130) diluted 1:250,000 and SuperSignal West Dura Substrate. Blots were imaged with a CCD camera and 1 second exposures.
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