GST 96-Well Detection Module for ELISA

Extracted from GST Gene Fusion System, GE Healthcare, 2014

The GST 96-Well Detection Module provides a highly sensitive enzyme-linked immunosorbent assay (ELISA) for testing clarified lysates and intermediate purification fractions for the presence of GST-tagged proteins (see Figs 4.1 and 4.2). Samples are applied directly into the wells of the plates, and GST-tagged proteins are captured by specific binding to anti-GST antibody that is immobilized on the walls of each well. Captured GST-tagged proteins are then detected with HRP/Anti-GST conjugate provided in the module. Standard curves for quantitation of tagged proteins can be constructed using purified recombinant GST, which is included as a control.

Each detection module contains reagents sufficient for 96 detections. Each plate is an array of 12 strips with eight wells per strip, such that as few as eight samples (one strip) can be assayed at a time.

The GST 96-Well Detection Module can also be used with antibody directed against a GST-tagged partner to screen and identify clones expressing the desired GST-tagged protein.

Sensitive detection of recombinant GST using the GST 96-Well Detection Module.

Fig 4.1. Sensitive detection of recombinant GST using the GST 96-Well Detection Module. Recombinant GST protein was prepared in 1× blocking buffer, and 100 µl volumes were applied directly to the wells of a GST 96-well capture plate. After 1 h binding at room temperature, the wells were washed in wash buffer and incubated with a 1:1000 dilution of HRP/Anti-GST conjugate for 1 h. Detection was performed using 3, 3',5,5'-tetramethyl benzidine (TMB) substrate, and the absorbance of each well was measured at 450 nm.

Fig 4.2. Screening of bacterial lysates for GST-tagged protein expression using the GST 96-Well Detection Module.

Fig 4.2. Screening of bacterial lysates for GST-tagged protein expression using the GST 96-Well Detection Module.

Each tagged protein is captured uniquely; therefore, if quantitation is required, prepare standards of recombinant GST protein and the tagged target protein (if available) using a dilution series from 1 ng/µl to 10 pg/µl in 1× blocking buffer. Include recombinant GST protein as a standard control in every assay.

Prepare fresh buffers daily.

Components of GST 96‑Well Detection Module

  • GST 96-Well Detection Plates (each well is coated with goat polyclonal anti-GST antibody, blocked, and dried)
  • Horseradish peroxidase conjugated to goat polyclonal anti-GST antibody (HRP/Anti-GST)
  • Purified recombinant GST standard protein

Additional reagents required for ELISA

PBS: 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3
Wash buffer: 0.05% Tween™ 20 in PBS (500 ml/96-well plate). Store at room temperature until needed.
Blocking buffer (1×): 3% nonfat dry milk in PBS with 0.05% Tween 20 (10 ml/96-well plate)
Blocking buffer (2×): 6% nonfat dry milk in PBS with 0.1% Tween 20 (5 ml/96-well plate)
Substrate  
Procedure
  1. Bring each test sample to a final volume of 50 µl with PBS.
  2. Add 50 µl of 2× blocking buffer to each sample.
  3. For screening, dilute the recombinant GST protein standard to 1 ng/100 µl in 1× blocking buffer.
  4. For quantitation, prepare a dilution series from 1 ng/µl to 10 pg/µl in 1× blocking buffer for both the recombinant GST protein and the target tagged protein (when available).
  5. Remove one 96-well plate from its foil pouch.

If using fewer than 96 wells, carefully remove the well strips from the holder by pushing up on the wells from below. Store unused well strips in the pouch with the desiccant provided.

  1. Pipette 100 µl of sample into each well.
  2. Incubate for 1 h at room temperature in a humidified container or incubator.
  3. Invert the plate and flick sharply to empty the contents of the wells.

Biohazardous material should be pipetted or aspirated into a suitable container.

  1. Blot the inverted plate or well strips onto a paper towel to remove excess liquid.
  2. Wash each well five times with wash buffer by inverting and flicking out the contents each time.
  3. Blot the inverted plate or well strips onto a paper towel to remove excess wash buffer.
  4. Dilute the HRP/anti-GST conjugate 1:10 000 (1 µl:10 ml) in 1× blocking buffer.

One 96-well plate will require 10 ml of the diluted conjugate.

  1. Add 100 µl of diluted HRP/anti-GST conjugate to each well and incubate for 1 h at room temperature in a humidified container or incubator.
  2. Empty the well contents and wash twice with wash buffer as previously described.
  3. Add soluble horseradish peroxidase substrate1 to each well and incubate according to the supplier’s instructions.

1 3,3',5,5'-tetramethyl benzidine (A450) and 2',2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) (A410) have been used successfully.

  1. Read plate absorbance in a microplate reader or spectrophotometer.

Materials

     
Related Links

Related Protocols