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Biological Assays

The types of assays available are  Enzyme-Linked Immunosorbent Assay (ELISA or EIA) and Protein (Bradford and DC/RC Lowery) Assays.  Many assays use the same principles to analyze samples and are optimized to meet the needs of the researcher.  The type of assay you choose is dependent on many factors including the cost,  how much volume, collection methods and type of sample is needed to answer your research question.  The core room where these assays are performed has the required supplies and equipment necessary to process the sample saving the researcher time and money.

The type of training available is:

  1. Operator Assisted – working with the researcher on the bench one on one throughout the assay or on a one time basis until trained and the Operator performs the assay Independently.
  2. Independent – researcher works on their own inside the core room after demonstrating competency with equipment and laboratory practice.
  3. Consulting – regarding any aspect of the technique or protocol the researcher needs assistance throughout the experiment, whether one time meeting or throughout the protocol including troubleshooting or optimizing a protocol with the operator.

Below is a:

  • Overview of how the assay works.
  • A list of the kits ran in the core for these assays.

 

 ELISAEnzyme-Linked Immunosorbent Assay (ELISA) technique is based on the antibody sandwich principle. they are plate-based assays designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones.  Other names as enzyme immunoassay (EIA) are also used to describe the same technology.  In an ELISA, an antigen is immobilized to a solid surface (plate) and then complexed with an antibody that is linked to any enzyme.  Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate (c0lor) to produce a measureable product.   The iMark Plate Reader is used to quantify this assay in the core (does not read Fluorescence labels – see Biology core website for a listing of fluorescence readers under instrumentation located in PSC).

 

 

Listed below are a few of the assays validated in the core.   Assays have been validated for multiple species as well as human saliva and blood samples with commercial kits to accommodate the researchers needs.  The requirements to run most kits are based on wavelength of reader and apparatus such as shakers and heated incubation shakers.  The equipment is designed for 96 well plate size.   Contact us if would also like information and consultation on what type of kit to use for your research. 

  • Cortisol
  •  Corticosterone
  • Testosterone
  • Estrodial
  • Oxytocin
  • Creatinin
  • Alpha Amylase
  • DHEA (Dehydroepiandrosterone)
  • BDNF (Brain Derived Neurotrophin Factor)
  • Alanine Transaminase Assay (ALT)
  • Insulin
  • Tissue Glycogen

 

 

lowry_vs_bradfordPROTEIN Assay is the determination of concentration of total concentration of protein in a solution. These assays are usually run in conjunction with RIA’s or ELISA’s when tissue extracts are analyzed.   A standard curve is created to determine the absorbancy of the sample which used in statistical analysis. Their are other types of protein assays using different wavelengths that can also be done in the core, please check to see what wavelength is available on the equipment to read your assay.  These assays are in kits including pre-made standards and dye.  The Geneysis 5 Spectrometer for cuvettes and iMark Plate Reader are used to measure these assays in the core.

 

 

 

The Protein Assays used in the core most often are:

  • cuvette BradfordBradford Protein assay, a colormeteric protein assay based on absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions the red form of the dye is converted into its bluer  form to bind to the protein being assayed.

 

 

 

  • (HDC/RC Lowry Protein method is best used when protein concentrations of 0.01 – 1.0 mg/mL and is based on the reaction of Cu+ produced by the oxiadation of peptide bonds, with Folin-Ciocalteu reagent.  The assay preferred for samples prepared with detergent or reducing agents.  The reagents for this assay are ordered when a request is made due to expiration times on them. 

 

 

LIGAND BINDING AND GTPgamma BINDING Assays using Tritium (H3) and Sulfur 35 (S35) has been performed in the lab.  The Ligand Binding assay was used to determine Kd (affinity) and Bmax for the (Mu Opoid Receptor) in rat brain membrane.  The procedure used 13 ml Polypropylene tubes for incubation followed using a 12 well manifold  on filtration paper transferred to a scintillation vial with scintillation fluid for quantification in the Tri-Carb Scintillation counter.   The GTP gamma assay was performed similarly.  Further information please contact the Core.