Problem 1：No signal
Possible Source	Test or Action
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.

 

Problem 2：High background
Possible    Source	Test or Action
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.            For phospho-specific antibodies: milk    contains casein which is a phosphor-protein; the phospho-specific antibody    will detect casein present in the milk causing high background. Use BSA as a    blocking reagent instead of milk.
The washing of unbound antibodies may be    insufficient	Increase the number and time of washes.
Your choice of membrane may give high    background	Nitrocellulose membranes are considered to    give less background than PVDF.
The membrane has dried out.	Care should be taken to prevent the membrane    from drying out during incubation

 

Problem 3：Multiple bands
Possible    Source	Test or    Action
Cell lines that have been frequently passaged    gradually accumulate differences in their protein expression profiles	Go back to the original non-passaged cell    line and run these samples in parallel.
The protein sample has multiple modified    forms in vivo such as acetylation, methylation, myristylation, phosphorylation,    glycosylation etc	Examine the literature and use an agent to    remove modifications where possible so that the protein runs at the expected    size.
The target in your protein sample has been    digested (more likely if the bands are of lower molecular weight)	Make sure that you incorporate sufficient    protease inhibitors in your sample buffer.
Unreported novel proteins or different splice    variants that share similar epitopes and could possibly be from the same    protein family are being detected	Check the literature for other reports and    also perform a BLAST search; use the cell line or tissue reported on the    datasheet.
Primary antibody concentration is too high	Try decreasing the concentration of the    primary antibody. Run a secondary antibody control (without the primary).
The antibody has not been purified	Try to use affinity purified antibody. This    will often remove non-specific bands.
The bands may be non-specific	Where possible use blocking peptides to    differentiate between specific and non-specific bands. Only specific bands    should be blocked (and thus disappear).
The protein target may form multimers	Try boiling in Laemmli buffer for 10 min    rather than 5 min to disrupt multimers.

 

Problem 4：Uneven white spots on the blot
Possible    Source	Test or    Action
Air bubbles were trapped against the membrane    during transfer or the antibody is not evenly spread on the membrane	Make sure you remove bubbles when preparing    the gel for transfer. Incubate the antibodies while agitating.

 

Problem 5：Black dots on the blot
Possible    Source	Test or    Action
The antibodies are binding to the blocking    agent.	Filter the blocking agent.

 

Problem 6：White bands on a black blot (negative of expected blot)
Possible    Source	Test or    Action
Too    much primary and/or too much secondary antibody.	Dilute the antibodies more.

 

Problem 7：Molecular weight marker lane is black
Possible    Source	Test or    Action
The    antibody is reacting with the molecular weight marker.	Add a blank lane between the molecular weight    marker and the first sample lane.

 

Problem 8：Band of interest is very low/high on the blot
Possible    Source	Test or    Action
Separation    is not efficient.	Change the gel percentage: use a higher    percentage for small proteins and a lower percentage for large proteins.

 

Problem 9：Smile effect on the bands
Possible    Source	Test or    Action
Migration was too fast.	Decrease the voltage while running the gel.
Migration was too hot (changing the pH and    altering the migration).	Run the gel in the cold room or on ice.

 

Problem 10：Uneven band size in lanes probed for the same protein
Possible    Source	Test or    Action
Gel    has set too quickly while casting and the acrylamide percentage is not even    throughout the gel.	Review the recipe of the gel and the addition    of TEMED to the gels, add some 0.1% SDS in water to the top of the migrating    gel while it sets to stop it from drying.

 

Problem 11：Uneven staining of the gel
Possible    Source	Test or    Action
Contamination    from bacteria	Keep antibodies at 4°C and use fresh buffers    covering the gel.
Not enough antibody volume	Make sure the membrane is covered with the    antibody and incubate while agitating.

 

Absin Bioscience Inc.

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