WB IFC ELISA IF(FAQs)-Updates-北京诺梵生物科技有限公司

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1.Old detection reagent or substrate kits

Use new detection buffer.

Avoid using buffers containing sodium azide, which may quench HRP signals.

3.Insufficient incubation with the primary antibody

Increase the incubation period with the primary antibody or perform overnight incubation at 4℃.

Do not over-wash the membrane - excessive washing may wash away the primary antibody. Try washing three times of 5 minutes.

5.Blocking buffer

Non-fat milk may mask antigens. Try to reduce the amount of milk in the blocking buffer and antibody solution or substitute it with different blocking reagents.

6.Incomplete transfer

Optimize the transfer conditions (e.g. time, electrical current). Reassure the completion of the transfer procedure before moving onto the next step

7.Over-use of antibodies

Use fresh antibody solution for the best performance.

8.Incompatible secondary antibody used

Use a secondary antibody which was rasied againt the host species of the primary antibody. Use positive controls to ensure that the secondary antibody is working properly.

9.Insufficient the primary antibody

Use higher concentrations of the antibody. Our protocol provides a guideline for antibody dilutions. Use positive controls to ensure that the primary antibody is working properly.

10.Difficult target proteins are presented

Try to use the most sensitive detection reagent.

11.Insufficient protein loaded into the gel

Increase the amount of sample for gel loading. Use 25-50ug cell/tissue lysate protein per lane. Load at least 25ug cell lysate protein per lane.

1.Over exposure

Decrease the exposure time.

2.Insufficient washing

Increase the number of washes with PBS to remove unwanted residual antibodies.

3.Insufficient blocking

Extend the blocking time or perform overnight blocking at 4℃.

4.Cross-reaction between blocking reagent and antibodies

Add Tween 20 (detergent) to the washing buffer or try different blocking buffer.

5.Non-specific binding of the antibody

Increase the blocking incubation period. Add 5% non-fat milk powder and Tween 20 to the blocking buffer.

6.The concentration of antibodies is too high

Dilute the antibody concentration further or incubate shorter.

1.Protein degardation or digested

Use protease inhibitors to improve the condition.

2.Different splice variants

Search information related to the expression of the target protein.

3.Protein modification

Search related documents or use some bioinformatic tools for modification prediction.

4.The secondary antibody concentration is too high

Decrease the incubation period or the concentration of the antibody. Run a lane with the secondary antibody only as control.

1.Incompatible secondary antibody

Choose a secondary antibody that is against the IgG of the host species in which primary antibody has been raised.

2.Insufficient incubation time with antibodies

Extend the incubation time.

3.Insufficient amount of antibody for the protein detection

Try to use higher concentrations of the primary antibody with a longer incubation time

4.Insufficient amount of target protein presented in the sample

Check information related to tissue specificity of the target protein. Use the most sensitive detection reagent.

5.Masked antigen

Use appropriate antigen retrieval method to break the protein cross-links formed by formalin fixation.

6.Insufficient deparaffinization

Refresh xylene solution and deparaffinize the sample sections for a longer period of time.

1.Autofluorescence or natural fluorescence existed in tissue

Before incubating with the antibody, check the tissue slide by a fluorescence microscope. If the fluorescence signal is detected, choose other detection systems, not the fluorescent method.

2.Endogenous biotin signal

Pretreat the tissue sample with unconjugated avidin if the biotin-avidin detection system is used.

3.Endogenous alkaline phosphatase activity

Pretreat the tissue sample with levamisole solution if AP is used as the label.

4.Endogenous peroxidase activity

Pretreat the tissue sample with 3% H2O2 before incubating it with the primary antibody.

5.Non-specific binding of the secondary antibody

Run a lane of control with only the secondary antibody.

6.The concentration of the antibody may be too high

Decrease the antibody concentration. '

High background/absorbance in negative control

1.Over incubation with the substrate

Try decreasing the incubation time to prevent signal over-development.

2.Substrate incubation is performed under the light

After adding substrate, put the plate in the dark immediately.

3.Reused plates

Use a new plate.

4.Substrate buffer is old

Use fresh substrate reagent.

5.The antibody concentration may be too high

Decrease the concentration of the antibody.

6.Insufficient blocking time

Extend the blocking time or use more/different blocking solution.

7.Contamination of the negative control

May be contaminated by other samples or reagent. Avoid overflowing among wells. Pipetting carefully.

8.Insufficient amount of washing for the plates

Make sure wells are filled up with wash buffer and ensure the complete removal of the residual solution from each well. Increase the number of washes.

Low absorbance value

1.Incorrect wavelength

Make sure that correct wavelength is set to measure the signal.

2.Insufficient incubation time for the substrate solutions

Optimize the incubation time until the plate has developed enough for ELISA reader.

3.Insufficient antibody used

Try to use more concentrated antibody. Incubate the antibody for the recommended amount of time

4.Target protein is not expressed in the sample

Search information related to the expression profile of the target protein. Increase the amount of sample used or use more sensitive detection reagents.

Poor duplication or reproducibility

1.Pipetting error

Be careful upon sample loading and avoid the presence of bubbles in wells.

2.Uneven coating

Check the coating procedure and use an appropriate ELISA plates.

No signal/Signal weak	l

1.Sample fixation destroys epitope

Try a different sample fixation method such as formalin or glutaraldehyde. Methanol may cause cell membrane to collapse.

2.Cell permeability is poor

Use 0.1 Triton X-100 for 15 min to permeabilize cells or increase time of incubation.

3.Insufficient wash

The slides be gently washed using PBS. Do not immerse in wash buffer for long periods or douse with distilled water.

4.The secondary antibody was exposed to light

Store the secondary antibody in the dark.

5.Insufficient secondary antibody used

Make sure secondary antibody is specific for primary antibody.

6.Blocking is too strong

Reduce blocking time.

7.Too few cells expressing protein

Try to use more cells in sample or use a stably transfected cell line.

8.Protein is not expressed in cells

Prepare a cell extract and check that target protein is present in cells by Western blots.

9.Antibody incubation time is too short

Extend the incubation time.

10.Antibody concentration is too diluted

A higher concentration is suggested.

High background or non-specific staining

1.Sample washed not enough

Wash thoroughly with PBS at the end of each step for 5 minutes. Repeat the process 5 times.

2.Incubation temperature is too high

Incubate samples at 4℃ overnight or at room temperature for 4 hours.

3.Secondary antibody is not specific

Check that secondary antibody is binding specifically. Run a secondary control without primary antibody.

4.Incubation period is too long

Incubate with primary/ secondary antibody for a short time.

5.The primary antibody is too concentrated

Dilute the antibody concentration.

6.Incorrect blocking buffers us, ed

Try to use a different blocking buffer such as 5% goat serum in PBS for 1 hour.

7.Overfixation of the test sample

Decrease fixation time.