Technical Information

General Procedure for Intra-Cellular Staining

  1. (a) Suspension cells: Transfer cells to a 15 ml conical tube and centrifuge at 1000-1200 rpm for 5 minutes. Discard media and wash cells 2X with 5 ml 1X dPBS. (b) Adherent cells: Trypsinize cells with Trypsin/0.25% EDTA. neutralize the trypsin with an equal volume of media once the cells are detached. Transfer cells to a 15 ml conical tube and centrifuge at 1000-1200 rpm for 5 minutes. Discard media and wash cells 2X with 5 ml 1X dPBS.

General Protocol for Human Interferon Alpha Cytopathic Effect (CPE) Assay

The antiviral-based Cytopathic effect (CPE) assay is amongst the most sensitive assays for interferon bioactivity, with often >100 fold more sensitivity than alternatives. Many combinations of cells and viruses can be used to measure interferon activity via CPE assay. Many researchers use the human lung carcinoma cell line A549 due to their ease of growth and relatively high sensitivity to all forms of human interfer­ons. The following is a summary of an A549/EMCV Cytopathic Effect Inhibition assay for Recombinant Human Interferon Alpha:

 

General Procedure for Cell Surface Staining

Materials:

  1. Appliable stimulated and un-stimulated cells
  2. 1X PBS pH 7.2
  3. Bovine Serum Albumin (BSA)
  4. Normal human serum
  5. 5 ml polystyrene tubes; BD Falcon Catalog No. 352052
  6. Isotype control: PE-Mouse IgG1 kappa; BD Pharmingen Catalog No. 554680

 

General Spike-Recovery Protocol for ELISA

The Spike and Recovery method is an important technique for analyzing and accessing the accuracy of ELISA and other analytical methods for particular sample types. It is used to determine whether analyte detection can be affected by the difference between diluent used for preparation and the experimental sample matrix.

 

Importance of ELISA Standard Curve

Introduction

The Enzyme-Linked Immunosorbent Assay (ELISA) is a highly sensitive procedure to quantify the concentration of an antibody or antigen in a sample. The estimation of the analyte concentration depends upon the construction of a standard curve. The standard curve is prepared by making serial dilutions of one known concentration of the analyte across a range of concentrations near the expected unknown concentration. The concentration of unknown samples is determined by interpolation which relies on a properly generated standard curve.

ELISA Data Analysis with Four-Parameter Curve Fit

Enzyme-linked immunosorbent assays (ELISA) are specific and highly sensitive procedures for identifying and quantifying analytes such as proteins in samples. This assay is based on the binding of a target molecule (analyte/antigen) to antibodies which recognize the compound. The presence of an antigen-antibody complex is detected using a secondary enzyme-conjugated antibody. Detection is obtained by addition of a substrate which yields a measurable product. Enzyme-linked immunosorbent assays are routinely used in many areas of biological research.

ELISA Troubleshooting Guide

Possible problem

Possible cause or experimental variables

Possible corrective action

Blank well absorbance too high Contaminated reagents or contaminated well Check the storage condition. Repeat with new reagents from the same lot if possible.

General Protein Handling Guide

Brief Guidelines for Freezing and Thawing Protein Samples

 

Disclaimer: These guidelines are intended for use in general with protein solutions, but the stability of individual proteins varies widely. The investigator must determine the proper storage and freeze-thaw conditions for each protein.

 

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