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Home > Technical Resources > Troubleshooting Guides

Troubleshooting guides

The guides below address common issues that may arise in your use of our products. They provide suggested troubleshooting solutions. If you have encountered any issues or require further assistance, please do not hesitate to contact us at [email protected].

ELISA troubleshooting

High Background 

Possible case

Solution

Non-specific binding of antibody

Replace another purified antibody or blocking buffer 

Plate are not be sealing incompletely

Make sure to follow the instruction strictly

Incorrect incubation temperature

Incubate at room temperature

Incubation time too long

Reduce incubation time

Substrate exposed to light prior to use

Keep substrate in a dark place

Substrate was contaminated 

Replace substrate. Substrate should be clean and avoid crossed    contamination by using the sealer

Contaminated wash buffer

Use a clean buffers and sterile filter

Improper washing

Increasing duration of soaking steps


Weak Signal 

Possible case

Solution

A reagent or a step of the procedure omitted by mistake

Check protocol and follow steps carefully

Antibody are not enough

Increase the concentration of the antibody

Reagent are contaminated

Use new one

Pipette are not clean

Pipette should be clean

Wrong incubation time or temperature

Check and follow protocol recommendations. Place plates in an incubator during incubation periods to avoid temperature   fluctuations (set to room temperature 25°C). 

Improper washing

Increasing duration of soaking steps


No Signal 

Possible case

Solution

Reagent are contaminated

Use new one

Sample prepared incorrectly

Make sure the sample workable/dilution

Antibody are not enough

Increase the antibody concentration

Wash buffer contains sodium azide

Use a new wash buffer and avoid sodium azide in it

HRP was not added

Add HRP according to the instruction


Poor Precision 

Possible case

Solution

Pipetting error

Dispense quickly and identically into the side of each well. Use     calibrated pipettes.

Incomplete washing

 

Make sure wells are washed adequately by filling the wells with wash buffer and all residual antibody solutions crossed well before washing.

Unclean wells

Inspect wells and remove debris prior to use. Wipe bottom of plate to remove any debris or fingerprints prior to reading


Poor standard curve

Possible case

Solution

Incorrect preparation of standard

Reconstitute standard as suggested on data sheet.

Capture Antibody did not bind

Use BT LAB ELISA plate in the kit

Inefficient washing

Be sure wash apparatus is working properly (i.e. distributing even volumes into each well). Be sure wells are empty after aspiration, yet be sure to fill wells in a timely manner.

Pipetting error

Dispense quickly and identically into the side of each well. Use calibrated pipettes.

Incorrect storage of components

Store all components as recommended on data sheet. Do not allow reconstituted reagents to stay at room temperature for excess time.


Western blotting troubleshooting

High background

Possible case

Solution

Blocking of non-specific binding might be absent or insufficient

Increase the blocking incubation period and consider changing the blocking agent, we recommend 3–5% non-fat dry milk, BSA, or normal serum for 1 h at room temperature. These can be included in the antibody buffers as well.

The primary antibody concentration may be too high

Titrate the antibody to the optimal concentration. Incubate for longer but in more dilute antibody (a slow but targeted binding is best).

The incubation temperature may be too high

Incubate membrane at 4°C.

The secondary antibody may be binding non-specifically or reacting with the blocking reagent

Run a secondary control without the primary antibody.

Cross-reactivity between the blocking agent and primary or secondary antibody

Add a mild detergent such as Tween 20 to the incubation and washing buffer.

The washing of unbound antibodies may be insufficient

Increase the number and time of washes.

The membrane has dried out.

Care should be taken to prevent the membrane from drying out during incubation

 

Weak or No signal

Possible case

Solution

The primary antibody and the secondary antibody are not compatible

Use a secondary antibody that was raised against the species in which the primary was raised (eg if the primary is raised in rabbit, use an anti-rabbit secondary).

Not enough primary or secondary antibody is bound to the protein of interest

Use a higher concentration of antibody or incubate longer (eg overnight) at 4°C.

There is cross-reactivity between the blocking agent and the primary or secondary antibody

Use a mild detergent such as Tween 20 or switch blocking reagent (ie commonly used blocking reagents are milk, BSA, serum or gelatin).

The primary antibody does not recognize the protein in the species being tested

Check the datasheet or perform a BLASTp alignment to see whether your antibody should react with the target protein. Run the recommended positive control.

There is insufficient antigen

Load at least 20–30 μg protein per lane, use protease inhibitors and run the recommended positive control.

The protein of interest is not abundantly present in the tissue

Use an enrichment step to maximize the signal (eg prepare nuclear lysates for a nuclear protein).

There is a poor transfer of protein to membrane

Check the transfer with a reversible stain such as Ponceau S. If proteins have not transferred effectively, check the transfer was not performed in the wrong direction. If using PVDF membrane, make sure that you pre-soak the membrane in methanol and then in transfer buffer.

Excessive washing of the membrane

Reduce the number or duration of washing steps.

Overuse of the primary antibody

Use fresh antibody as the effective concentration is lowered upon each use.

The secondary antibody is inhibited by sodium azide

Do not use sodium azide together with HRP-conjugated antibodies.

The detection kit is old and the substrate is inactive

Use fresh substrate.

 

Splotchy background

Possible case

Solution

Uneven antibody distribution

Ensure the membrane is agitated evenly by placing on a rocker or shaker during incubation.

Insufficient amount of incubation buffer

Ensure enough solution is present during each incubation period to fully submerge the membrane and allow it to float freely in the solution.

Air bubbles present during transfer

Gently remove all air bubbles from transfer "sandwich" before transfer is started. This can be done by using a roller or clean glass rod/Pasteur pipette.

Membrane problems

Ensure membrane is wetted thoroughly according to the manufacturer's protocol.

Handle membranes with extreme care.

Use forceps or wear gloves when handling membrane.

Aggregation of HRP conjugate

If using HRP, filter conjugate to remove HRP aggregates. Use a fresh sample of HRP conjugate.

Contamination of reagents

Filter buffers before use to remove contaminant. Make fresh buffers and re-run Western Transfer.

 

Non-specific bands

Possible case

Solution

Antibody concentration too high

Reduce concentrations of antibodies, particularly of primary antibody.

Too much protein loaded on gel

Reduce the amount of sample loaded on gel.

 

Air bubbles present during transfer

Gently remove all air bubbles from transfer "sandwich" before transfer is started. This can be done by using a roller or clean glass rod/Pasteur pipette.

 

 

 

 

Signal from chemiluminescent substrate too strong

Reduce the length of time the blot is exposed to film.

Reduce the concentration of the substrate.

Shorten incubation time of membrane with substrate.

Completely remove substrate after incubation period.

Decrease the concentration of antibodies, particularly HRP- and AP-conjugated antibodies.

 

IHC troubleshooting

Lack of staining

Possible case solution

Lack of antigen.

Check protein expression by in situ hybridization (in some rare cases translation may be blocked even though mRNA is detected).

Antibodies do not work due to improper storage.

Follow storage instructions on the datasheet. In general, aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Store aliquots in a manual defrost freezer (-20 to -70°C) and avoid repeated freeze-thaw cycles.

Inactive primary or secondary antibodies.

Test reporter system independently to assess reagent viability. 

Inadequate tissue fixation.

Try increasing the fixation time or try a different fixative.

Tissue overfixation.

Reduce the duration of the immersion or post-fixation steps. If immersion-fixation cannot be avoided (e.g., collection of postmortem tissues or biopsies in pathology lab), antigens may be unmasked by treatment with antigen retrieval reagents.

Incompatible secondary and primary antibodies. 

Use a secondary antibody that will interact with the primary antibody. For example, if the primary antibody was raised in rabbits, use an anti-rabbit secondary antibody. 

Antigen was destroyed before incubation with the primary antibody. 

If quenching of endogenous peroxidase was done prior to the addition of primary antibodies, block peroxidase after incubation with the primary antibody. 

Epitope altered during fixation or embedding procedure.

Try restoring immunoreactivity through various antigen retrieval techniques. Embed tissue at 58°C or below.

Antigen retrieval was ineffective.

Increase the time of treatment or change the treatment solution.

Reagents omitted or used in wrong order.

Repeat staining and confirm that correct reagents are used and are added in the correct order.

 

Inappropriate staining

Possible case solution

Fixation method is inappropriate for the antigen.

Try a different fixative or increase the fixation time.

Antigen retrieval may be inappropriate for this antigen or tissue.

Try different antigen retrieval conditions.

Electrostatic charge of the antigen has been altered.

Try adjusting the pH or cation concentration of the antibody diluent. 

Delay in fixation caused diffusion of the antigen.

Fix tissue promptly. Try a cross-linking fixative rather than organic (alcohol) fixative.

 

Cell/Tissue morphology destroyed

Possible case solution

Antigen retrieval methods are too harsh.

Empirically determine the conditions that preserve tissue morphology while restoring the immunoreactivity of the antigen.

Tissue sections falling off slide (more common with frozen sections).

Increase fixation time. Empirically determine an additional or alternative fixative. Use freshly prepared, adequately charged slides.

Tissue section appears torn or folded. Air bubbles under section.

Re-cut sections using a sharp blade, or ignore damaged areas when analyzing the results.  

Poor resolution of tissue morphology.

Cut thinner tissue sections. Ice crystals may have destroyed morphology of frozen sections.

Underfixation has physically damaged the tissue or cells during the staining process.

Increase fixation time and/or add a post-fixation step. Increase the fixative/tissue ratio. Cut smaller pieces of tissue for more thorough immersion-fixation.

Autolysis of tissue leading to staining of necrotic debris. 

Increase the fixation time, ratio. Consider using cross-linking fixative. 

 

High background

Possible case solution

High concentration of primary and or secondary antibodies.

Titer antibody to determine optimal concentration needed to promote a specific reaction of the primary and the secondary antibodies.

Hydrophobic interactions of the antibody and proteins in the tissue.

Lower the ionic strength of the antibody diluent (particularly, monoclonal antibodies respond well to reducing the salt concentration).

Non-specific binding of primary and/or secondary reagents to tissues.

Use blocking step just prior to primary antibody incubation (we use 1% BSA with 10% normal donkey serum, or non-fat dry milk is another option).

Non-specific binding of secondary antibody.

Use an antibody that has had cross-reactive IgG species removed (absorbed against sample species).

Tissue dried out.

Avoid letting the tissue dry during the staining procedure.

Reagents sticking to old or poorly prepared slides.

Start over with freshly prepared or purchased slides.

Background due to ionic interactions.

Increase the ionic strength of the diluent buffer.

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