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SCHOTT Nexterion Slide H is a slide surface ideally suited for the printing of protein microarrays, as it is ideally suited for the covalent immobilization of peptides and proteins such as antibodies, antibody fragments, enzymes, or receptors. For many protein microarray applications Nexterion® Slide H has proven to be a very attractive alternative to the commonly used nitrocellulose coated slide, especially where low background, or slide transparency are important considerations. Since its introduction, the slide coating has also been successfully used with amino-modified oligonucleotides, and has become the slide of choice for printing amino-linked glycan microarrays. Carbohydrate arrays are a rapidly growing area of microarray research and Nexterion® Slide H is an excellent choice for use in the rapid screening of carbohydrateprotein interactions. The permeable, polymer coating has a large immobilization capacity, and helps to preserve the native three-dimensional structure of complex bio-molecules, thus maintaining conformation and functionality. Nexterion® Slide H produces excellent signal-to-background ratios and an exceptionally wide dynamic range compared to conventional “two-dimensional” coatings through a unique combination of low non-specific binding characteristics, and high probe loading capacity. Even very low intensity signals, such as those obtained from low-abundance analytes, or weakly expressed genes can be reliably detected and quantified on Nexterion® Slide H. The robust coating matrix is fully compatible with commercial microarray printers and scanners. Simple and robust protocols are available making Nexterion® Slide H easy-to-use.
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PepTalk – The Protein Information Week. San Diego, CA. January 12-15, 2004
Nexterion Slide H Offers Excellent Protein Stability, Low Nonspecific Binding and High Sensitivity for Protein Microarraying Applications
Jo Ann Kraycer, Brenda Baggett, Luis Burzio ([email protected]), and Sam Conzone. SCHOTT Nexterion. A Division of SCHOTT North America, Inc.
400 York Avenue, Duryea, PA 18642-2036. (570)457-7485
Abstract
The marked structural differences between DNA and proteins has lead to the need for surface coatings that are suitable for protein arraying in terms of stability, deterrence of nonspecific binding and sensitivity. Proteins should ideally be immobilized in an environment that elicits minimal interaction, and provides suitable hydration. A hydrogel coating provides such an ideal environment for protein immobilization. Hydrogels have a high affinity for water, and can thus preserve the hydration layer that surrounds immobilized proteins. Further, the permeable, 3-dimensional structure of a hydrogel is well suited for the preservation of native protein structure, and can be designed to exhibit low nonspecific binding and good loading capacity, which together can result in excellent sensitivity. Here we present data from NexterionTM Slide H, a proprietary hydrogel coating that maintains protein stability, offers low nonspecific binding, and high sensitivity for many protein microarraying applications. Protein stability was assessed by immobilizing enzymes directly onto Nexterion Slide H, other polymer coated slides and silane (epoxy and aldehyde) coated substrates, and then measuring enzymatic activity spectrophotometrically. The results demonstrate that for both Horse Radish Peroxidase and Alkaline Phosphatase, 5-20 times more protein activity was maintained after spotting onto Nexterion Slide H, as compared to the silane coated substrates, and is comparable to products currently in the market. In addition to the excellent retention of protein stability, Nexterion Slide H has good loading capacity, which together provide for high signal intensities during protein microarray analyses. The chemical composition of the Nexterion Slide H polymer matrix allows for very low nonspecific binding even in the presence of “sticky” proteins (i.e. insulin) and complex protein mixtures (i.e. serum or cell extracts). This low nonspecific binding characteristic only requires chemical deactivation of the active groups within Nexterion Slide H and does not require blocking with protein solutions (e.g., Non-fat milk or Albumin) which can interfere with probe/target interactions. Nonspecific binding was characterized by incubating deactivated Nexterion Slide H and silane coated substrates with Cy3® labeled insulin and serum, followed by scanning. Chemically deactivated Nexterion Slide H substrates yielded 50 times less signal due to nonspecific binding than silane coated substrates after deactivation. Results were similar or better when compared to the leading 3D surface. Such low non-specific binding will result in low background intensities during protein microarray analyses. Finally, it will be shown how the combination of excellent protein stability, loading capacity and low non-specific binding for Nexterion Slide H can offer excellent sensitivity for protein microarray applications. Results from antibody, antigen, and complex protein mixture microarray assays are presented to highlight the excellent sensitivity and versatility provided by Nexterion Slide H.
Experimental Procedures
•The general experimental procedure used for protein
microarray assays on Slide H is shown below.•The manufacturer-supplied protocols were followed for assays conducted on non-slide H surfaces.
Dilute probes in print buffer (Antibodies, Antigens, Protein Mixtures)
Print Probes using BioChip Arrayer (Perkin Elmer)
Immobilized for 1 hour
Chemically Inactivate (Blocked) for 1 hour
Incubated with target solution for 1 hour
Washed PBS+0.5%Tween® (3X)
Scanned on Axon 400B at 600PMT
YNexterion Slide H
Capture Antibody
Labeled Target
Anti-IgG was spotted at different concentrations. The labeled target was IgG-Cy5® at 10μg/ml.
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5
10
15
20
Slide H Leading 3D Slide
S/B
Detection of IL-1α in normal serum. Serum was diluted to 25% and the cytokine was detected using a 10μg/ml solution of monoclonal Anti-IL1-α-Cy5®.
Y
Nexterion Slide H
Complex Protein Mixture Spotted
Labeled Antibody
The capture antibody (Anti-IgG) was printed at different concentrations. The target solution was 25% normal serum. The labeled detection antibody was Anti-IgG-Cy5® at 10μg/ml.
Y
YNexterion Slide H
Capture Antibody
Detection Antibody
Target Antigen
Images at various Probe Concentrations (g/ml)
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250100
50
25
10
5
Slide H Leading 3D Slide
Y
Nexterion Slide H
Antigen Probe
Labeled Antibody
The detection of simple antigen probes can theoretically be hindered by the hydrogel matrix, especially if the protein probe is small. In this case, Insulin (MW 5800) was printed onto the slides at different concentrations and detected with Anti-insulin-Cy5® at a concentration of 10μg/ml .
The Signal/Background (S/B) was determined for the 50g/ml probe concentration.
Excellent Sensitivity can be Achieved with Nexterion Slide H Using Various Protein Microarraying Assays
•The detection of protein targets with arrays is typically achieved using antibodies or complex protein mixtures as probes. The most typical assays are exemplified below.•When compared to the leading 3D Slide, Slide H exhibited superior S/B at a particular probe concentration, thus resulting in better sensitivity for detecting proteins of different abundance (e.g.,IgG versus IL1-)
Conclusions
1. Slide H is retains 5-20 times more protein activity that conventional silanized slides
2. Low nonspecific binding is achieved with chemical deactivation and without the need for protein-containing blocking solutions.
3. Slide H is highly sensitive in many different assays with 2-8 times more signal than the leading 3D Slide under the same assay conditions.
4. Slide H is a robust and versatile surface for protein microarraying, allowing for long print runs, short incubation times and highly specific interactions.
Antibody Capture Assays Sandwich Assays
Antigen Probe Assays
Complex Antigen Detection
Serum-Cy3
Nor
mal
ized
Flu
ores
cent
Sig
nal
EAB PBA
Aldehyde
Epoxy
Leading 3D Slide
Slide H
EAB PBA
Insulin-Cy3® Serum-Cy3®
Nexterion Slide H Provides Exceptionally Low Nonspecific Binding After Chemical Deactivation or Blocking
Scanned Images after Incubation*
Insulin-Cy3
Quantitative Comparison
* Slides were scanned using an Axon 400B scanner at full laser power and 600PMT
AldehydeEpoxy
Leading 3D SlideSlide H
Images at various Probe Concentrations (g/ml)
1000
500
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5025
10
Slide HLeading 3D Slide
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250
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Anti- IgG Concentration (µg/ ml)
S/B
Slide H Leading 3D Slide
Nexterion Slide H Provides Excellent Protein Stability
•The Slide H surface chemistry is tailored to preserve the native structure and activity of printed proteins.
•Enzymes are especially susceptible to adverse environments, and were thus used to characterize the protein stability properties of Slide H.
•Enzymes were printed on various slide surfaces at a concentration of 1mg/ml. The slides were then incubated for 1 hour at room temperature and 50% relative humidity before assessing the activity of the immobilized enzymes spectrophotometrically.
Printing Washing Incubation Activity Measured
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Slide H Aldehyde Epoxy Leading 3DSlide
% R
elat
ive
Act
ivit
y
Retained Activity of Alkaline Phosphatase
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100
Slide H Aldehyde Epoxy
% R
elat
ive
Act
ivit
y
Retained Activity of Horseradish Peroxidase
Leading 3DSlide
Nexterion Slide H has a Robust Chemistry that Enables Long Print Runs, Short Incubation Times and Use of Complex Protein Target Solutions
3. There is little effect on the S/B result when the complexity of a target solution is increased. In this example, the S/B from Anti-IgG:IgG interactions is nearly unaffected by the presence of other proteins, indicating the specificity of the reaction as well as the low nonspecific binding characteristics of the Slide H surface.
2. Slide H allows for short target incubation times. In this examples Anti-IgG was printed and detected with IgG-Cy5® at times ranging from 5 to 120 min.
1. The stability of the active chemistry in the Slide H coating allows for long print runs without loss of binding capacity. Here Streptavidin-Cy5® was printed at time zero and after 8 hours of incubation at room temperature and 50% relative humidity.
0200400
600800
100012001400
25 50 100
Cy-labeled Streptavidin Probe Concentration (g/ml)
S/B
Leading 3D Slide Slide H
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50
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350
5 15 30 45 60 90 120
Target Incubation Time (min)
S/B
050
100150200
250300350400
IgG IgG+HSA IgG+STP IgG+HSA+STP
IgG+Serum
Cy5-Labeled Target Composition
S/B
The normal serum concentration of IL1-α is 10pg/ml
0
20
40
60
80
100
EAB PBA EAB PBA
•High background can result from the nonspecific binding of target proteins to active and
inactive chemical groups that are present on the coated surface.
•Various slides were incubated for 1 hours at room temperature with a Cy3 labeled target solution (insulin or serum) after chemical deactivation with a 50mM Ethanolamine buffer (EAB) or blocking with a phosphate buffer containing 0.5% Tween and 2%BSA (PBA).
0 h
r
8 h
r
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r
8 h
r
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r
8 h
r
0 h
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8 h
r
0 h
r
8 h
r
0 h
r
8 h
r
0
100
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0 250 500 750 1000
Anti- IgG (g/ ml)
S/B
Slide H Leading 3D Slide
0
2
4
6
Slide H Leading 3D Slide
S/B
