Вопросы и ответы Сведения о конфигурации
What surfaces are available for biochemical assays?
Biochemical assay microplates are available in three formats: standard biochemical, enhanced biochemical, and streptavidin-coated.
Вопросы и ответы Сведения о конфигурации
Can I capture biotinylated proteins through immobilized streptavidin?
Yes. Biotinylated peptides, proteins, and nucleic acids can be captured via immobilized streptavidin. Protocols are available for streptavidin immobilization on standard biochemical assay microplates. Commercially available streptavidin-coated microplates are also available.
Вопросы и ответы Сведения о конфигурации
Why don't I observe any immobilization of my protein?
Suboptimal immobilization pH or insufficient protein concentration are the most common causes of low protein immobilization. For any new protein target, we recommend testing immobilization across a range of pH values. For some proteins, we have observed that capturing efficiency is highest when using a pH of 0.5 and 1 unit below the isoelectric point of the protein. Generally, a protein concentration of 50 µg/mL works well, but for some proteins, higher concentrations may be required.
Вопросы и ответы Сведения о конфигурации
What is the difference between the Epic standard and enhanced biochemical microplates?
Both the standard and enhanced Epic biochemical microplates immobilize target proteins via primary amine coupling. The enhanced microplates are user-activated and provide enhanced sensitivity, particularly for assays with very small response windows. The enhanced Epic microplates contain a spacer arm chemistry which may enable better positioning and presentation of the immobilized protein for binding site availability.
Вопросы и ответы Сведения о конфигурации
What are the advantages of the self-referencing technology in biochemical assay microplates?
Relative to adjacent well referencing, self-referencing technology delivers data with lower variability and minimizes the number of additional reference wells required.
Вопросы и ответы Сведения о конфигурации
What is the compound molecular weight detection limit for biochemical assays?
The smallest molecular weight compound that can be detected is a function of the size of the immobilized protein and ratio of binding. For a 30 kDa protein, the smallest analyte we have detected binding in a 1:1 ratio is ~150 Da. The larger the immobilized protein, the larger the analyte must be to detect binding.
Вопросы и ответы Сведения о конфигурации
What is a basic assay flow for a biochemical assay?
The basic assay flow for a biochemical assay is 1) target immobilization (1 hour to overnight); 2) wash to remove unbound protein; 3) sensor equilibration; 4) baseline read; 5) analyte addition and mixing; 6) final read.
Вопросы и ответы Сведения о конфигурации
How much protein is required for protein immobilization?
The amount of protein required for immobilization is somewhat protein dependent, but good results can generally be obtained using a concentration of 50 µg/mL.
Вопросы и ответы Сведения о конфигурации
How much thermal equilibration time is required for biochemical assays?
We recommend at least a 20 min equilibration time at the temperature of the Epic instrument prior to initiating baseline acquisition.
Вопросы и ответы Сведения о конфигурации
What is the correlation between the Epic response and mass density?
As a general guideline, 1 pm of response corresponds to a protein density of ~3-5 pg/mm2
Вопросы и ответы Сведения о конфигурации
Can I use Epic to determine stoichiometry?
Yes. In a biochemical assay, it is possible to determine if the binding signal levels are consistent with 1:1 binding versus a higher stoichiometry. By measuring the amount of protein immobilized, one can calculate a theoretical maximum binding signal for an analyte. If the observed signal in an assay is significantly larger than the maximum theoretical signal, this could indicate either greater than 1:1 binding stoichiometry or compound aggregation.
Вопросы и ответы Сведения о конфигурации
Can Epic Technology be used to determine kinetic constants of ligand binding?
Because Epic Technology does not utilize flow cells, measurements of kinetics (kon, koff) will be diffusion-limited.
Вопросы и ответы Сведения о конфигурации
Can Epic Technology be used to identify promiscuous or aggregating compounds?
Yes. In a biochemical assay, it is possible to determine if the binding signal levels are consistent with 1:1 binding versus a higher stoichiometry. By measuring the amount of protein immobilized, one can calculate a theoretical maximum binding signal for an analyte. If the observed signal in an assay is significantly larger than the maximum theoretical signal, this could indicate either greater than 1:1 binding stoichiometry or compound aggregation.
Вопросы и ответы Сведения о конфигурации
Can I use part of an Epic biochemical assay microplate?
In general we do not recommend using a portion of the microplate with the intent to use the remainder at a later time. Biochemical assay microplates are coated with a pre-activated surface chemistry that has an open pouch life of ~8 hours.