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Western Blot troubleshooting

Sensitivity of Western Blot detection is down to 10 ng of protein.

When working with proteins of VERY low abundancy or if wanting to improve overall detection we recommend ECL Advance Western Blot detection reagent from AmershamBiosciences.
Results obtained with this reagent are presentend using our commercial antibodies. anti-RbcL antibody.
Please, note that both, primary and secondary antibodies are used in the dilution 1: 50 000. (with standard ECL reagent recommended dilutions with this product are in the range: 1: 2000 to 1: 20 000).

Problems:

High background signal
Strange/Non-specific bands on the blot
Low sensitivity
Is a right band detected?
Uneven results with lots of spots

Results not reproducible

Often caused by not strict following the procedure (varied incubation and agitation times,speeds and temperature), amount and buffer volumes used for washes etc. If large number of membranes needs to be processed, more automated equipment can be used, like Processor Plus, check with Amersham Biosciences.

Basic verification of results

First step: gel stained with Coomasie after the transfer to check if most of the proteins have been transfered succesfully from the gel.
Second step: staining membrane after the transfter with Panceau Red (stain is washed with dest. water, and can be followed by incubations with primary antibody afterwards), to check quality of transfer and assure that proteins are actually on the membrane. Sensitivity of Panceau stain is not so high, 1 ug of protein can be detected this way. Not optimal transfer conditions can result in proteins passing through membrane druing transfer. This can result in no signal detection of Western Blot.
Third step: Review your Western Blot protocol, e.g. protocols involving incubations over few days are not necessary. Wetsern Blot procedure can be completed within couple of hours.

Each primary-secondary antibody pair might require slight adjustment of the current procedure.

High background signal

Two main types of background signal:

patchy, uneven spots all over the membrane

unspecific bands detected

are discussed below.

If you work with chicken IgY antibodies, please, check our protocol for Western Blot using IgY

High background signal - blocking problems

Secondary antibody reacts with blocking reagent. Test it by:
Omit primary antibody in your procedure and test with secondary only. If background appears change either secondary antibody or blocking reagent
Blocking not good enough (importance of this step is often underestimated).
Nonfat dry milk (5%) is most commonly used. Presence of detergent in blocking solution, like Tween 20 at 0.05 % is also very important. Extend blocking time to at least one hour at RT.
Note: blocking overnight at 4°C might decrease efficiency of blocking, since the detergent might not be so effective in lower temperature. Also other contaminants present in serum or total IgY can stick to the membrane and increase a background signal. This will depend from quality of a primary antibody sample and will vary between different antibodies.
- Disguises some antigens
- Incompatible with avidin/streptavidin (milk contains biotin)
- Complex carbohydrates in milk will absorb out antibodies that recognize carbohydrate determinants.
- Some IgY antibodies might recognize milk proteins.
- 10 % nonfat dry milk might block more efficiently however in some cases so well, that no bands of interest will be seen
Problems with backgorund while using nonfat dry milk can be often solved by trying BSA instead.

Alternative Blocking reagents:
- BSA (3%)
- Serum
  Some animals from which blocking serum has been obtained may have developed antibodies to the antigen in question. If this is the case, they may bind to the antigen and prevent the primary antibody from binding.
- When protein blocking solution can not be used try Tween 20, used at:
  0,2-0,5 % in PBS, blocking for 1 hour at RT. Primary antibodies incubated in 0,25 % Tween20/PBS for 1 hour at RT. Note: this method might need to be optimized for different primary antibodies. Also, Tween20 will not block efficiently binding sites on molecular weight markers./li>
  Reference about no-protein containing blocking buffers: Batteiger et al. 1982 PubMed reference
  Test different blockers and adjust your protocols accordingly.
High background - other reasons
Too high concentration of either primary or secondary antibody. Titrate the concentration of antibodies used in the assay. Concentration of the secondary antibody is usually well above 1:10 000
Another batch of secondary antibody is used and varies in activity. Dilute secondary antibodies 5 or 10 x more than it has been used before. Different batches of secondary antibodies even from the same producer can vary in activity.
Primary antibody might not be specific or contain impurities. Try another antibody or an affinity purified antibody.

Note: however, affinity purification might not be an ultimate solution, if antigen specific antibodies recognize epitopes specific to other proteins. In this case more attention should be paid to blocking step.

High background can result form presence of antibodies which might bind to cognate or related epitopes of proteins preset in the tested sample. This type of contamination is specific. Try to use affinity purified antibodies if possible.
Membrane not suitable for the application - test another membrane. The way the membrane is wetted in a first step is important.
Always handle membrane with care e.g. use gloves, use agitation during washes and incubations.
For consistent results, use the same times and speeds while agitating the membrane.
Consider flat containers for membrane handling.
For blocking serum from for example goat is used. Secondary antibody used comes from same animal species. This will casue increased background problems.

Strange/Non-specific bands on the blot

"Ghost bands at HMW" can be a result of oxidation of a part of reducing agent. Cysteines are not protected and this can lead to aggregation. Therefore after boiling of the sample before loading on the gel, reducing agent should be added one more time.
Negative bands on the blot: either the amount of target protein on the membrane or the amount of primary antibody used is exceeded. More antibody added results in less signal detected. Adjust antigen or antibody concentrations.
Streaks - solid particles in the sample
Smears - glycosilated proteins can give this effect

Non-specific bands

Too short washes. Increase washing time.
Pre-adsorb primary antibody (overnight, 4C) with tissue extract which lacks your protein of interest; You can use PVDF membrane after gel elcetrophoresis/transfer/Panceau Staining and excission of a band/area containing your target protein
Possibility in case of using anti-peptide antibodies: antibodies against carrier protein (KLH, BSA or others) might recognize epitopes of other proteins present in the extract.
Anti-carrier antibodies can be removed by:
- incubating serum/IgY sample with a carrier protein in solution (0.1 % (w/v) or spotted on PVDF membrane
- passing serum/IgY sample through the column with bound carrier protein. Use flow through fraction in further experiments.

Is a right band detected?

It is often difficult to rule out, if a detected chemiluminescent signal on a Western Blot comes from a target protein under investigation.
Following methods can help:

1. Perfomr a peptide competition assay, in case on anti-peptide antibody.
2. In this case (if possible) doing Western Blot with another antibody against your target, which will bind to other epitopes on a target protein, might help to provide additional evidence and support final conclusion.
3. Western Blot on the samples from a mutant lacking protein of interest.

Low sensitivity

Even though antibodies are a very sensitive tool, there is a limit to their detection. Often optimisation of Western Blot conditions can help to solve this problem.

Can be improved by:

Reducing non-specific background.
Amplification of the existing signal, by for example by biotinylation of primary antibodies.
Usage of a stronger signal, e.g. using chemiluminescence instead of a colorimetric method.

Some reasons for it:

Too short reaction time between antibodies and antigen. Increase the incubation time.
Primary antibody concentration is too low. Use more of the primary antibody.
Not correct primary antibody. Not all antibodies are working in all applications. The antibody might not recognize denatured epitope (if antibodies are used on the material after SDS page). Try detection on native protein instead, after electrophoresis without SDS and sample boiling.
Too low amount of the antigen of interest in the cell extract. Load more of the antigen on a gel if possible, by using for instance cell extracts from conditions or cell lines, where protein of interest is expressed in higher levels. Use more sensitive detection system. Make sure that latest extracton methods have not been changed.
Not right gel system used. For some antibodies pre-cast gels might work much better than for the poured gels. We have seen the antibodies which did not give any signal using self made gels, to a good signal on pre-cast gels, by the same antibody.
Too much of substrate for the enzyme in the system, results in fading bands. Decrease the amount of substrate used.
Substrate for secondary antibody is too old. Check expire date on your reagents.
Secondary antibody inhibited by sodium azide. Check your system, do not use sodium azide together with HRP conjugated antibodies. Inhibition usually happens when primary antibody is used in dilutions < 1 : 1000. Use alkaline phosphatase system, which is not sensitive for sodium azide.
Secondary antibody does not bind to the primary antibody. Spot the primary antibody on the membrane. Block. Incubate with the secondary antibody and check if the signal will appear, what means that the secondary antibody binds to the primary. If possible use affinity purified secondary antibodies.
Too extensive washing. Check your washing time with the protocol, adjust washing time, remove detergent from washing buffer.
Transfer to the membrane was not sufficient. Check your transfer efficiency by staining the membrane after the transfer with Ponceau S before blocking (destaining with water). Good info about problems with staing with Ponceau Try another membrane, note that PVDF might not work with high molecular weight proteins. Also make sure that gel concetration is suitable for molecular weight of the protein unders investigation. Another solution: use reagents allowing detection using antibodies directly in gel, omitting transfer step (Pierce).
Check your power supply and make sure it works correctly.
Effects of some parameters for efficiency of Western Blot transfer:
- SDS present in transfer buffer will increase the mobility of proteins out of the gel and due to giving a negative charge to the protein will help to maintain it in the soluble state.
- SDS present in the transfer buffer will reduce protein binding to the nitrocellulose membrane due to decreased hydrophobicity of the protein. Note that SDS can also affect antigenicity of some proteins.
- Alcohol present in the transfer buffer will decrease protein mobility out of the gel. It will also reduce pore size of the gel, while it will improve binding to nitrocellulose. Alcohol will also remove SDS from proteins and increase their hydrophobicity.
- If protein of interest does not have a negative charge - transfer efficiency will be decreased
- Field strength (V/cm) - if too low the mobility of proteins out of the gel will be decreased. Proteins will not be completely transfered from the gel. If too high - proteins will pass through the membrane without binding.
- Gel thickness - for thinner gels protein mobility out of the membrane will be increased. Draw back is lower protein capacity of such gels and more possible detection problems. If working with low molecular proteins using too thick gels can cause lack of the bands...
High molecular weight proteins: might not be transfered completely if methanol was present in the transfer buffer. Change membrane to nitrocellulose, omit methanol from transfer buffer, while adding SDS and increasing field strength.
Low molecular weight proteins: might pass through the membrane during transfer. Shorten transfer time, use double layer of membrane.
Highly hydrophobic membrane proteins: might not be unfolded enough to expose epitopes antibodies has been produced against. Use 2-8 M urea in the gels and sample buffer.
In some cases, storing primary antibodies (at 4°C or -20°C) in solution with blocking reagent for further use might result in decreased primary antibody activity.

Uneven results with lots of spots

Contamination. Check that all your buffers are free from bacterial contamination and particulates. Check that deionized water is coming from a well maintained supply.
Not enough of the solution during incubation or washing. Make sure that your membrane is well emerged in the solution, which is moving gently across the membrane.
During incubation air bubbles became trapped on the surface of the blot. Next time, gently remove all air bubbles.

Examples of "bad gels" and some explanations for those results: Gel goofs

Unanswered questions? feel welcome to e-mail: joanna@agrisera.se

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