Creative Biolabs has strong expertise and extensive experience in affinity and kinetics measurement with cell lysate. We can satisfy any specific demand with our label-free and high efficiency affinity Surface Plasmon Resonance (SPR) and Bio-Layer Interferometry (BLI) technologies.

Background

Various methods have been developed to analyze the affinity of interested protein with other proteins and components in crude cell lysate. One classic method is affinity purification, which needs the purified protein of interest to carry out the followed binding analysis step. However, Creative Biolabs can provide label-free protein affinity detection in crude cell lysate based on SPR technique with anitbody arrays, without the need of highly-purified protein samples. (Figure 1, left paenel).

Figure 1. SPR sensorgram of a lysate prepared with E. coli which expresses 6xHis-tagged 14-3-3 protein, the sample was injected over a sensorchip with immobilized Ni2+ while the chip was regenerated with 0.3 M imidazol (left); Overview of reduction of non-specific adsorption from cell lysate with peptide SAMs (right). (Mol. Biotechnol., 2006)

There are two problems in affinify measurement with cell lysate: 1). Bulk of effort, which can be overcomed by adjusting the reflectivity of running buffer with that of the lysate; 2). Non-specific adsorption, which can be reduced by the hydrophilicity and low electrostatic charge of the surface chemistries (Figure 1, right panel). The optimized system enables high-throughput analysis of diverse proteins with minimal sample volume (200 μL). The antibody array on the gold chip was placed into an SPR instrument (Figure 2).

Figure 2. The surface chemistries of the fabricated antibody arrays. (Anal. Chem., 2005)

This system is applicable for, but not limited to:

• Cell lysate.
• Cell phenotyping.
• Food analysis.
• Discovery of new biomarkers.
• Immunological disease diagnostics.

Affinity Measurement for Cell Lysates

Creative Biolabs can provide custom affinity and kinetics measurements with cell lysate based on label-free and high-throughput SPR and BLI technologies. All the data analysis will be performed and documented. Please feel free to contact us for a detailed quote.

Other optional Antibody Affinity Measurement Services:

• Cell Line Affinity Measurement
• Affinity Measurement Services for Specific Antibodies
• Membrane Protein Affinity Measurement
• Affinity Measurement Services for Bivalent Antigens
• Affinity Measurement Services for Dimers/Polymers
• Affinity Measurement Services for Small Molecules
• Affinity Measurement Services for Various Ligands and Analytes
• Biacore System
• ProteOn System
• Octet system

Published Data

• The Effect of Receptor Phosphorylation on the Binding Between IRS-1 and IGF-1R as Revealed by SPR biosensor

Fig. 3 Sensorgrams for the capture of IGF-1R from the NIH-3T3 cell line with or without overexpression of IGF-1R by the immobilized anti-IGF-1R antibody. (Minghui Huang, 2001)

Here, the researchers developed a receptor binding test based on surface plasmon resonance (SPR) biosensor technology. This binding assay was used to study the interaction between the insulin-like growth factor-1 receptor (IGF-1R) and its intracellular substrate protein, insulin receptor substrate-1 (IRS-1). Membrane fragments containing protein receptors can be captured from cell lysates by modifying the sensor surface with anti-IGF-1R (α subunit) monoclonal antibodies. IGF-1R is immobilized on the surface of the sensor and has the ability to bind to its intracellular substrate. The results of SPR detection showed that the tyrosine phosphorylation of IGF-1R induced by its extracellular ligand, insulin-like growth factor-1, caused the receptor to bind to IRS-1 10 times faster than that of the unactivated receptor.

FAQ

1. What is the advantage of using SPR technique with antibody arrays for measuring affinity and kinetics in cell lysates?

   The advantage of using the SPR technique with antibody arrays for measuring affinity and kinetics in cell lysates lies in its ability to provide real-time, label-free analysis. This method allows for the simultaneous monitoring of multiple interactions, making it highly efficient for screening the binding affinity and kinetics of various biomolecules within complex mixtures like cell lysates. Additionally, the use of antibody arrays enables the specific detection of target molecules, even in the presence of other competing substances, which is crucial for accurate biophysical characterization of molecular interactions.

2. How do antibody arrays enhance the detection capabilities of the SPR technique for cell lysate analysis?

   Antibody arrays enhance the detection capabilities of the SPR technique by providing a high-throughput platform that can simultaneously test multiple interactions. This is particularly useful in cell lysate analysis where numerous potential binders are present. Each spot on the array can be functionalized with different antibodies, allowing for the detection and quantification of various analytes from the cell lysate. This multiplexing capability not only saves time and sample but also improves the throughput and data quality by enabling comparative studies across different interactions under the same experimental conditions.

3. Can the SPR technique detect low-affinity interactions in cell lysates using antibody arrays?

   SPR technique equipped with antibody arrays is capable of detecting low-affinity interactions in cell lysates. This capability is due to the high sensitivity of the SPR sensors and the specific binding properties of the antibodies on the arrays. By optimizing experimental conditions such as flow rate and antibody concentration, SPR can effectively measure even transient or weak interactions, providing valuable insights into the dynamic range of molecular interactions within cell lysates.

4. What are the sample preparation requirements for analyzing cell lysates with SPR and antibody arrays?

   Sample preparation for analyzing cell lysates with SPR and antibody arrays typically involves clearing the lysate of cellular debris through centrifugation or filtration to prevent sensor fouling and maintain the integrity of the sensor surface. The lysate may also need to be diluted to optimal concentrations to reduce matrix effects and ensure accurate measurement of binding kinetics and affinity. Proper preparation ensures that the SPR measurements are both reliable and reproducible.

5. How does temperature affect the measurements of affinity and kinetics in cell lysates using SPR?

   Temperature can significantly affect the measurements of affinity and kinetics in cell lysates using SPR. Most biochemical interactions are temperature-dependent, influencing the binding affinity and rate constants. SPR experiments are typically conducted at controlled temperatures to mimic physiological conditions and ensure consistent results. Variations in temperature can lead to alterations in the conformation and dynamics of the biomolecules, thereby affecting the interaction data obtained.

6. What is the role of the regeneration step in SPR analysis with antibody arrays?

   The regeneration step in SPR analysis with antibody arrays is crucial for removing bound analytes from the antibody surfaces without damaging the integrity of the antibodies. This step ensures that the array can be reused for multiple cycles of binding and analysis, which is particularly important for comparative studies and high-throughput screening. The choice of regeneration solution and conditions depends on the stability of the antibody-antigen interaction and the nature of the analyte.

7. How does the flow rate impact SPR measurements with antibody arrays?

   The flow rate in an SPR assay with antibody arrays impacts both the association and dissociation phases of the binding interaction. Higher flow rates can enhance mass transport to the sensor surface, reducing the time it takes to reach equilibrium and potentially increasing the association rate. However, too high a flow rate may also disrupt weak interactions. Conversely, a slower flow rate can improve the accuracy of dissociation measurements but may increase the time required for experiments. Optimizing the flow rate is essential for obtaining accurate kinetic data.

8. What types of analytes can be detected in cell lysates using SPR with antibody arrays?

   SPR with antibody arrays can detect a wide range of analytes in cell lysates, including proteins, peptides, nucleic acids, and small molecules. The versatility of the antibody arrays allows for the customization of the sensor chips to target specific molecules of interest, making it a powerful tool for analyzing complex biological mixtures and conducting biomarker discovery, drug target validation, and basic research in cellular and molecular biology.

Resources

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