Figure 3. No cross reactivity of Pro-Detect rapid GST assay strips with indicated crude lysates and purified proteins. As shown in the figure there was no cross reactivity of the Pro-Detect rapid GST assay strips with proteins endogenous to mammalian, bacterial, sf9 cells (lanes 1-5) or with any other non-specific tag proteins (lanes 7-19). In addition, even using 100% of Minimum Essential Medium with 10% FBS there was no cross reactivity detected. CHO and Hela cell lysates were prepared using nitrogen cavitation method in buffer a containing 10 mM Hepes, 150 mM KCl and 1 mM Mg(OAc)2 and used at 1 mg/ml. HEK 293 lysates were prepared in Pierce IP Lysis Buffer (Cat. No. 87787) and tested at 1 mg/ml. BL21 lysates were prepared using B-PER Bacterial Protein Extraction Reagent (Cat. No. 90084) following manufacturing instructions and diluted 4-fold using Pro-Detect dilution buffer to a final concentration of about 185 µg/ml. Pure proteins such as GST, MBP, SUMO and Fc tag proteins were used at 0.1 µg/ml whereas rest of the smaller tag containing proteins (lanes 13-19) were used at 10 µg/ml. Wherever necessary, all the dilutions were carried out using Pro-Detect dilution buffer in a total volume of 100 µl in a 96-well microtiter plate. Individual Pro-Detect rapid GST assay test strips were placed in each of the wells for 15 minutes. Appearance of a red line at the test line (T) was captured by either scanning the strips or taking a picture using a cell phone.
While there are variety of methods available to identify the presence or absence of a protein of interest, those methods can be laborious, time consuming and expensive. Thermo Scientific Pro-Detect Rapid Assay Kit and Pro-Detect Rapid Competitive Assay Kit enables one to determine if protein expression and purification methods are successful based on the detection of expressed tagged-proteins. These lateral-flow, dipstick format kits offer a simple and time saving alternative that allows for monitoring at all phases of protein expression and purification in real time. Just dilute the sample, dip a test strip in the sample, and get a visual qualitative result in 10–15 minutes.
Download the application note to get technical details of the assays from an R&D expert.
Highlights
- Fast and easy—obtain visual qualitative results in 10–15 minutes, without the need for special equipment
- Sensitive—detect proteins or antibodies in cell culture media, lysate or purified protein down to the ng/ml level
- Compatible—unaffected by typical concentrations of detergents and salts
- Reliable—results are comparable to the standard qualitative assays for protein detection such as western blotting and ELISA
- Specific—no cross-reactivity with other targets
- Cost effective—save money by preventing unnecessary downstream analysis
Applications
- Identify if protein expression is happening in the absence of induction
- Optimize protein expression conditions by determining optimal dose of inducer and optimal time of induction
- Rapidly identify elution fractions containing protein of interest.
Featured product data
Figure 1. The Pro-Detect rapid assay kit is a sandwich lateral flow assay. The gold-conjugated capture antibody specific to the analyte is embedded in the conjugated pad at the bottom of the strip. Additionally, detection antibody specific to the analyte is embedded at the test line, while a control antibody is embedded in a control line at the top of the strip (A). Upon application, the capture antibodies, bound to any present analyte, travel the length of the membrane and are resolved on the test and control lines, displayed visually by red bands (B). If no test band displays, then the sample is negative (B), while the display of both the test line and the control line indicates the presence of analyte in the sample (C).
Figure 2. The Pro-Detect competitive assay is a competitive lateral flow assay. The targeted analyte is immobilized on the membrane as 3 distinct test lines. A control antibody is immobilized as the control line. The gold-conjugated capture antibodies, specific to the analyte, are embedded in the conjugated pad (A). In a negative result where no analyte in the sample is present or concentration of analyte is below detectable levels, the gold-conjugated capture antibodies will bind to the analyte embedded on the test lines and form 3 visible red test lines (B). In a positive test where the sample contains analyte, the gold-conjugated antibodies embedded in the conjugated pad will bind to the available analyte in the sample and thus will not bind to the analyte immobilized at the test lines. As a result, the test lines do not show up as red lines. and competing for detection at the test line(s). The concentration of the analyte in the sample is inversely related to the number of test lines appearing on the strip (C). In both positive and negative tests, the gold-conjugated capture antibodies will bind to the control antibodies at the control line.
| Assay type | Target | Ideal range of detection* | Note |
|---|---|---|---|
| Sandwich assays | DYKDDDK-His | 0.1-10 µg/mL | The lateral flow assay will be able to detect outside of this range; however, intensity of the bands will be lower at concentrations higher and lower than the working range. It is not recommended to use at concentrations less than 0.01 μg/mL. |
| SUMO | |||
| GST | |||
| MBP | |||
| Human Fc | |||
| Rabbit Fc | |||
| Mouse Fc | |||
| Competition assays | His | 4-20 µg/mL | The competitive lateral flow assays can detect down to 1 μg/mL, but loss of band intensity may be less distinguished. As there is no upper limit, the lateral flow strip will continue to show positive results at concentrations greater than 20 μg/mL, visualized by complete loss of test lines (test lines closest to the sample will disappear first). It is not recommended to use at concentrations less than 1 μg/mL. |
| DYKDDDK | |||
| HA | |||
| Strep | |||
| AVI | |||
| Myc | |||
| V5 |
* Proper sample dilution is essential for optimal results. Concentration ranges are based upon the concentration of the tagged protein of interest in the sample.
We examined the performance of the Pro-Detect rapid assay in the presence of various concentrations of detergents and salts that are commonly used in protein expression and isolation. Table below summarizes our estimates of each assay tolerance levels for the tested detergents and salts.
Concentrations listed refer to the actual concentration in the protein sample. Pro-Detect rapid assay strips were tested with 0 µg/mL (negative control) or 0.1 µg/ml of a positive control protein for a sandwich type assay and 10 µg/ml of a positive control protein for a competition type of assay. All additives were added to PBS. The stringent conditions that did not interfere with the tests were listed in the table.
| Assay type | Target | Nacl | Urea | TX 100 | SDS | NP40 | EDTA | Gly-cerol | KCl | CHAPs | RIPA | BPER | MPER |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Sandwich assays | DYKDDDDK – His (Double Tag) | 1.5M | 0.4M | 1% | 0.20% | 1% | 7.5mM | 10% | 1.5M | 1% | 90% | 90% | 90% |
| SUMO | 1.5M | 0.4M | 1% | 0.20% | 1% | 7.5mM | 10% | 1.5M | 1% | 90% | 90% | 90% | |
| GST | 1.5M | 0.4M | 1% | 0.20% | 1% | 7.5mM | 10% | 1.5M | 1% | 90% | 90% | 90% | |
| MBP | 1.5M | 0.4M | 1% | 0.20% | 1% | 7.5mM | 10% | 1.5M | 1% | 90% | 90% | 90% | |
| Human Fc | 1.5M | 0.4M | 1% | 0.20% | 1% | 7.5mM | 10% | 1.5M | 1% | 90% | 90% | 90% | |
| Rabbit Fc | 1.5M | 0.4M | 1% | 0.20% | 1% | 7.5mM | 10% | 1.5M | 1% | 90% | 90% | 90% | |
| Mouse Fc | 1.5M | 0.4M | 1% | 0.20% | 1% | 7.5mM | 10% | 1.5M | 1% | 90% | 90% | 90% | |
| Competition assays | His | 0.5M | 0.4M | 1% | 0.20% | 1% | 5mM | 10% | 0.5M | 1% | 90% | 90% | 90% |
| DYKDDDDK | 0.25M | 0.4M | 1% | 0.20% | 1% | 5mM | 10% | 0.25M | 1% | 90% | 90% | 90% | |
| HA | 0.5M | 0.4M | 1% | 0.20% | 1% | 5mM | 10% | 0.5M | 1% | 90% | 90% | 90% | |
| Strep II | 0.5M | 0.4M | 1% | 0.20% | 1% | 5mM | 10% | 0.5M | 1% | 90% | 90% | 90% | |
| AVI | 0.5M | 0.4M | 1% | 0.20% | 1% | 5mM | 10% | 0.5M | 1% | 90% | 90% | 90% | |
| Myc | 0.25M | 0.4M | 1% | 0.20% | 1% | 7.5mM | 10% | 0.25M | 1% | 90% | 90% | 90% | |
| V5 | 1.5M | 0.4M | 1% | 0.20% | 1% | 7.5mM | 10% | 1.5M | 1% | 90% | 90% | 90% |
Western blotting is a widely used and accepted tool to identify a specific protein in a complex mixture. However, it is a time-consuming and labor-intensive process, with many preparative steps and time intervals to manage. The Pro-Detect rapid assays are able to provide comparable accuracy but with much shorter processing time (10-15 minutes) and less hands-on steps.
Figure 4. GST protein detection: Comparison of Pro-Detect Rapid GST Assay with Coomassie dye R-250 based protein staining method and with a Chemiluminescent based western detection.
Left panel: Indicated concentrations of a purified GST protein was separated on a 4-12% SDS-PAGE, and then stained with Imperial stain (Cat. No. 24615) for 1 hr followed by destaining with several washes with water over a period of 3 hrs.
Middle panel: Indicated concentrations of a purified GST protein was separated on a 4-12% SDS-PAGE, and then transferred onto a nitrocellulose (Cat. No. 88018) membrane using the Pierce Power Blotter (Cat. No. 22834) and 1-Step Transfer Buffer (Cat. No. 84731). The membranes were blocked with Starting block T20 (Cat. No. 37543) and incubated with antibodies against GST (Cat. No. MA4-004), followed by incubation with Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP Conjugate (Cat. No. 34265) at a concentration of 10 ng/mL. Chemiluminescent detection was performed following a 5-minute incubation with SuperSignal West Pico PLUS chemiluminescent substrate (Cat. No. 34580). Signal was captured at various times on a iBright Imager (Cat. No. A32752). Results shown above was captured at a 10 sec exposure.
Right Panel: Indicated concentrations of a purified GST protein were diluted with ProDetect Sample dilution Buffer in a total volume of 100ul in a 96 well microtiter plate. Individual ProDetect Rapid GST assay strips were placed in each of the wells for 15 min. Appearance of a red line at the Test line (T) was captured by either scanning the strips or taking a picture using a cell phone. Note that when protein concentration is expected to be higher than the upper limit of the linear range of detection which is typically about 1ug/ml, a false negative result appears at the Test line as a faded signal due to a phenomenon called hook-effect. Under these conditions it is recommended to dilute the same further 10-100 fold to perform the assay in the linear range.
Figure 5. DYKDDDDK tag protein detection: Comparison of Pro-DetectRapid DYKDDDDK Competitive Assay with Coomassie dye R-250 based protein staining method and with Chemiluminescent based western detection.
Left panel: Indicated concentrations of a purified DYKDDDDK-tagged recombinant protein was separated on a 4-12% SDS-PAGE, and then stained with Imperial stain (Cat. No. 24615) for 1 hr followed by destaining with several washes with water over a period of 3 hrs.
Middle panel: Indicated concentrations of a purified DYKDDDDK-tagged recombinant protein was separated on a 4-12% SDS-PAGE, and then transferred onto a nitrocellulose (Cat. No. 88018) membrane using the Pierce Power Blotter (Cat. No. 22834) and 1-Step Transfer Buffer (Cat. No. 84731). The membranes were blocked with Starting block T20 (Cat. No. 37543) and incubated with antibodies against DYKDDDDK epitope (Cat. No. PA1-984B), followed by incubation with Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP Conjugate (Cat. No. 31430) at a concentration of 10 ng/mL. Chemiluminescent detection was performed following a 5-minute incubation with SuperSignal West Pico PLUS chemiluminescent substrate (Cat. No. 34580). Signal was captured at various times on a IBright Imager (Cat. No. A32752). Results shown above was captured at a 2 sec exposure.
Right Panel: Indicated concentrations of a purified DYKDDDDK-tagged recombinant protein was diluted with ProDetect Sample dilution Buffer in a total volume of 100ul in a 96 well microtiter plate. Individual ProDetect Rapid His competitive assay strips were placed in each of the wells for 15 min. Disappearance of a red lines at the Test lines (T) indicating the presence of His tag proteins in the sample was captured by either scanning the strips or taking a picture using a cell phone.
Pro-Detect Rapid Assay Kits and Pro-Detect Rapid Competitive Assay Kits
Lateral-flow assay for antibody isotyping
The Pro-Detect Rapid Antibody Isotyping Kit is a lateral-flow assay with high sensitivity for rapid determination of the immunoglobulin class, subclass, and light chain type of monoclonal antibodies. Two different formats of rapid isotyping kits are available. These include dipstick format and cassette-based format.
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