Troubleshooting for Common Lab Applications

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General Lab Techniques: washing & pipetting

ELISA TroubleShooting

IHC Troubleshooting

General Lab techniques

Washing Techniques:

Multi-channel Pipette or squirt bottle             

Ensure that each prong of the pipette is dispensing properly or that the squirt bottle has enough pressure. Empty the contents of the well and fill well with volumes of wash buffer indicated. Decant the wells and invert and blot on a clean paper toweling. Repeat this process as indicated in the insert. After the last decanting, remove any remaining wash buffer by inverting and blot it on a clean paper toweling. Do not allow wells to sit dry, move to the next step as indicated in the insert. Note: numbering the strips prior to washing and decanting in a clean sink is useful in the event that strips become loose while decanting.

Manifold dispenser or autowasher        

Ensure that there is an appropriate vacuum supply. Check all cannulae or prongs for proper dispensing and aspirating. Empty the wells by aspirating or decanting. Fill each well the appropriate volume indicated in the insert. Aspirate the wells completely. Do not over aspirate the wells by allowing the aspiration aparatus to sit in the wells after the buffer has been removed. Repeat this process as indicated in the insert, not allowing the wells to sit dry after the last aspiration.

Pipetting Techniques:

Ensure that the pipette that you are using is calibrated and that the pipette tip is seated properly and securely on the pipette. Set the pipette to the desired volume and pre-rinse with sample or reagent. Draw the reagent up slowly, allowing the pipette to return to the up position slowly. Wait a moment to allow the reagent to reach volumetric equilibrium in the tip. Dispense the reagent slowly to the first stop and then gently to the second stop. Hold the pipette at this position until it is removed from the reservoir or well to avoid drawing the reagent back up. As you remove the pipette, draw the tip up the side of the reservoir or well to release any liquid that may be on the outside of the tip.

ELISA Troubleshooting

Many factors can affect the performance of an ELISA.

  • Read the instructions carefully prior to starting. This will help avoid unnecessary errors.
  • Use good laboratory practices as well as calibrated equipment.
  • Use clean reservoirs and a clean lab space and change pipette tips between each reagent.
  • Run all standards and samples in duplicate as this will improve accuracy.

Poor Precision:

  • Incomplete washing: ensure that apparatus is working correctly and that wells appear dry after aspiration.
  • Unequal mixing of reagents: Ensure that reagents are mixed adequately.
  • Contamination: Be sure to use clean reservoirs and change pipette tips between each reagent.
  • Pipetting error: use good lab techniques and ensure pipette is calibrated properly.

Standard Curve:

  • Improper standard preparation: Ensure that standards are reconstituted appropriately and that dilution instructions are adhered too.
  • Incomplete washing: ensure that apparatus is working correctly and that wells appear dry after aspiration.
  • Unequal volumes added to well: check that pipette is calibrated and that pipette tips are seated properly.
  • Ensure that blank and NSB values have been subtracted from the net ODs.

High Background:

  • Ensure that the plate was washed correctly. Review washing techniques above.
  • Adhere to all incubation times and temperatures. High backgrounds can result from extended incubation periods or incubation at higher than recommended temperatures.

 Drift

  • Bring all reagents to room temperature prior to use. 
  • Prepare reagents prior to running the assay or as directed in the kit insert.
  • Avoid interrupted assay set-up.

Edge Effect

  • Be aware of temperature variations or drafty areas when incubating the plate in ambient conditions. Placing the plate under an air conditioning or heating vent may cause uneven color development.

Color Development

  • Ensure all reagents are added at the correct volumes and at the correct times in the assay. Adhere to correct incubation times.
  • Read plate immediately after stop solution is added unless otherwise indicated in the kit insert.
  • Was the substrate prepared correctly and added
Data Reduction
  • For assistance on generating a standard curve, please visit our protocol section

IHC Troubleshooting

 No staining

Problem Possible Solution(s)
The primary antibody and the secondary antibody may not match. It is recommended that the secondary antibody be against the species in which the primary antibody was raised (e.g if the primary antibody is raised in mouse, use anti-mouse secondary antibody).
The antibody may not be suitable for IHC procedures which reveal the protein in its native (non denatured) form. Test the antibody on a non-denatured and denatured western blot to make sure the antibody is recognizing non-denatured antigen.
The secondary antibody was not stored in the dark. It is recommended to avoid exposing the secondary antibody to light.
Not enough primary antibody is binding the protein of interest. Dilute the antibody less.
incubate for longer at 4°C.
The protein of interest is not abundantly present in the tissue. Run a positive control.
Utilizing an amplification step can assist in amplifying the signal. 
Use a lower dilution of primary antibody.
The protein of interest is a nuclear protein and the antibody cannot penetrate the nucleus. To facilitate penetration of the nucleus, add a permeabilizing agent to the blocking buffer and the antibody dilution buffer.
Deparafinization may be insufficient Deparaffinize sections longer.
Use freshly prepared xylene.
Fixatives such as formalin and paraformaldehyde may be modifying the epitope the antibody recognizes. Use antigen retrieval method to unmask the epitope.
Fix for less time.
The PBS buffer may be contaminated with bacteria that has damaged the protein of interest. Use a preservative such as 0.01% azide in the PBS antibody storage buffer.
use fresh sterile PBS.

 High background

Problem Possible Solution(s)
Blocking of non-specific binding may be insufficient. Increase the blocking incubation period to 30 min for sections and 1 hour for cell culture. Use a blocking agent such 10% normal serum for sections and 1-5% BSA for cell culture.
The primary antibody concentration may be too high. Titrate the antibody to a more optimal concentration, incubate for longer using more dilute. This will provide a slower but more specific binding.
The secondary antibody may be binding non-specifically. Run a secondary control without primary antibody.
Incubation temperature may be too high. Incubate tissue sections or cells at 4°C.
Tissue not adequately washed. Wash extensively with PBS between all steps
Endogenous peroxidases are active. Use enzyme inhibitors such as Levamisol (2 mM) for alkaline phosphatase or H2O2 (0.3% v/v) for peroxidase.
Fixatives such as formalin and paraformaldehyde are too strong and may have modified the epitope the antibody recognizes. Change antigen retrieval methods.
Decrease the incubation time with the antigen unmasking solution.
Too much substrate was applied. Reduce substrate incubation time.
The chromogen reacts with the PBS present in the cells. Use a Tris buffer to wash sections prior to incubating with the substrate.
Pemeabilization has damaged the membrane and removed the membrane protein. Remove permeabilizing agent from your buffers.

 

Non-specific staining

Problem Possible Solution(s)
The concentration of the primary or secondary antibody may be too high.
Try decreasing the antibody concentration and/or the incubation period. Compare signal intensity against cells that do not express the target.
The primary antibody is raised against the same species as the tissue stained (e.g rat primary antibody tested on rat tissue). When the secondary antibody is applied, it binds to all the tissue since it was also raised against that species.
Use primary/secondary antibodies raised against a different species than your tissue.
Endogenous peroxidases are active. Use enzyme inhibitors such as Levamisol (2 mM) for alkaline phosphatase or H2O2 (0.3% v/v) for peroxidase.
The sections/cells have dried out. Keep tissue sections and cells at high humidity and do not let them dry out.