Now that the genome has been sequenced, researchers are directing their attention to large-scale studies of proteins including mapping of protein: protein interactions. Such studies require automated methods to overcome bottlenecks associated with protein purification for high-throughput assays. Integrated Lysis and Affinity Purification (iLAP®) plates represent a rapid and automation-compatible system for simultaneous cell lysis and affinity purification of histidine-tagged recombinant proteins.
The HIS-Select iLAP 96-well plates offer an ideal format for economical high-throughput screening and automation. The patent-pending iLAP plates are coated with cell lysis reagents and a HIS-Select nickel chelate matrix, allowing for cell lysis, protein capture, and assay of a histidine-tagged protein in a single well (Figures 1, 2, and 3). There is no need to harvest cells from culture; hence, automation is as simple as placing an E. coli culture that is expressing a target protein of interest on the deck of a liquid handler and executing the program.
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Automation Resources
Automated methods have been developed and validated for HIS-Select iLAP plates. The following resources are available for download:
Applications
Automation of recombinant protein purification using HIS-Select iLAP plates is suitable for a variety of applications including:
- Colony screening
- Direct quantitation of bound proteins by standard methods such as BCA assays
- ELISA
- Protein: protein interaction assays
Features & Benefits
| Rapid Procedure |
96 samples can be processed from cell lysis through recombinant protein purification in under 3 hours. |
| Enhanced Productivity |
Walk-away automation of cell lysis and recombinant protein purification allows you to concentrate on other aspects of your work. |
| Simplified Handling |
The optimized coating of lysis reagents allows for the extraction of soluble proteins directly from growing cells eliminating labor-intensive steps such as centrifugation and cell disruption. |
| Economical for Screening Applications |
In addition to lysis and purification, bound protein can be subjected to ELISA and quantitation assays in a single iLAP plate. |
| High Specificity |
The unique affinity matrix coating provides highly specific interactions with histidine-tagged proteins yielding proteins with greater than 90% purity. |
| High Binding Capacity |
Nickel chelate matrix has a binding capacity of greater than 4 µg/well. |
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| HIS-Select iLAP HC Nickel Coated Plates |
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| Product # |
Product Name |
Package Size |
| H9412 |
HIS-Select iLAP HC Nickel Coated Plate |
1 ea
5 x 1 ea |
| Supporting Reagents |
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| Product # |
Product Name |
Package Size |
| HS0100 |
HIS-Select Wash and Elution Buffer Kit
Includes 500 mL Wash Buffer and 250 mL Elution Buffer |
1 KT |
| H5413 |
HIS-Select Wash Buffer |
500 mL
1 L |
| H5288 |
HIS-Select Elution Buffer |
250 mL
500 mL |
| S3401 |
Sample Buffer, Laemmli 2x Concentrate |
6 mL |
Gel Analysis of Recombinant Protein Purification Using the Automated iLAP Method
Figure 1. SDS-PAGE Analysis of Purified Recombinant Proteins.
The automated Sciclone method was used to purify a target histidine-tagged protein from 100 µL of an E. coli culture. The purified protein was eluted with 250 mM imidazole. 20 µl samples were reduced and denatured for analysis by SDS-PAGE. The gel was then stained with EZBlue™ gel staining reagent (G1041). Lanes 2-12 correspond to wells A1, B2, C3, D4, E5, E6, E7, E8, F10, and G11 from the iLAP plate. Only the target protein of interest is detected on the gel. (A) Analysis of Samples from Random Wells. All 96 wells of the iLAP plate were loaded with E. coli culture, and samples were subjected to SDS-PAGE. (B) Cross Contamination Analysis. E. coli cells expressing a protein of interest were dispensed into odd numbered wells of the iLAP plate, all even numbered wells of the plate correspond to a negative control.
Specificity Analysis of Recombinant Protein Purification Using the Automated iLAP Method
Figure 2. Analysis of Protein Purity Achieved with iLAP Plates.
The automated Sciclone method was used to purify a target histidine-tagged protein from 100 µL of an E. coli culture. The purified protein was eluted with 250 mM imidazole. 20 µl samples were reduced and denatured for analysis by SDS-PAGE. Lanes 2-12 correspond to wells A1, B2, C3, D4, E5, E6, E7, E8, F10, and G11 from the iLAP plate. Only the target protein of interest is detected on the gel. (A) ProteoSilver Stained Gel. The gel was stained with ProteoSilver™ gel staining reagent (PROT-SIL1). (B) Western Blot Analysis. 1 µg of purified protein was transferred to the blot.
Direct Quantitation of Bound Protein
Figure 3. Total Protein Quantification.
Target protein was processed using the automated iLAP method for protein purification and subsequent BCA Assay Setup. (A) Standard Curve. BSA standards were serially diluted and setup for BCA analysis using the Sciclone ALH 3000. Following a 2-hour incubation, absorbance measurements were taken and the standard curve was plotted. Linear regression analysis was used to calculate the equation of the line and the R2 value. (B) Plate Map Quantifying Bound Protein. Total protein concentrations were calculated from standard curve shown in Figure 3A. Mean values and coefficients of variation were calculated for the whole plate.
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