Western Immunoblotting Troubleshooting Guide

• Western Blotting & Immunoprecipitation Products
• Protocols
• Changes to Recommended Western Blotting Protocols

Problem: High Background or Extra Bands

General background is high or nonspecific bands appear after a short exposure of blot to film. Please be conscientious of the fact that motif antibodies, post-translational modifications, and splice variants may result in multiple banding.

Watch these clips on how to overcome a high signal problem

High Background

Lysate preparation

Freshly prepared samples result in fewer nonspecific and degradation bands, and therefore yield a cleaner blot. In general, tissue extracts tend to contain more background bands and degradation products than cell line extracts due to connective tissue. Using fresh, sonicated and clarified tissue extracts may lessen background. Samples should always be lysed in appropriate buffers that include protease inhibitors and phosphatase inhibitors for phospho-targets (Phosphatase Inhibitor Cocktail (100X) #5870, Protease/Phosphatase Inhibitor Cocktail (100X) #5872, Protease Inhibitor Cocktail #5871). We recommend the use of Cell Lysis Buffer (#9803) or RIPA Buffer (#9806), which contains stronger detergent, for sample preparation and protein concentration quantification. SDS loading buffer (#7722 or #7723) may be used for whole cell lysis without protein quantification. Chaps Cell Extract Buffer (#9852) is used to prepared cytoplasmic cell fractions and is useful for studying caspase signaling.

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Membrane

Use only high quality nitrocellulose membrane (Nitrocellulose Sandwiches #12369). Pore size 0.2 µm is generally recommended; membranes with a pore size of 0.45 µm are not recommended for proteins smaller than 30 kDa. Nylon membranes are not recommended for western blotting. Block for 1 hr at room temperature in 5% nonfat dry milk in TBST.

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Primary antibody dilution and/or incubation

Incubate primary antibody overnight at 4°C in TBST at the recommended dilution with the recommended blocking agent (either 5% BSA or 5% nonfat dry milk). For individual antibodies please consult the product datasheet for recommended dilution buffer.

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Inadequate washing

Washing for less time than the recommended 3 x 5 min in TBST is common, and can result in high background. We recommend that washes be timed to ensure accuracy. This applies to washing steps after both primary and secondary antibody incubations.

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Secondary antibody dilution and/or incubation

Some secondary antibodies bind nonspecifically to proteins in cell extracts. To assess the quality of a secondary antibody, perform a blot (through to film exposure) without primary antibody. Serial dilutions of the secondary antibody can be performed on blots with the same cell extracts and primary antibody to optimize secondary antibody concentration. Always incubate the secondary antibody in 5% nonfat dry milk in TBST for 1 hr at room temperature, diluting the secondary antibody in BSA yields higher levels of background bands. CST™ secondary antibodies have already been optimized and do not require titration.

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Detection reagent

Chemiluminescent detection requires that high purity water be used to dilute concentrated reagents. Use only purified water with organic and inorganic impurities removed. We recommend using RODI (reverse osmosis deionized) water. Additionally, ensure that the membrane is never allowed to dry out during antibody incubation process prior to detection reagent exposure.

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Exposure time

Long exposure times of more than 30 seconds lead to increased background signal. To avoid long exposure times, it is important to use cell lines or tissues with adequate protein expression levels and use recommended primary antibody incubation conditions (see primary antibody dilution and/or incubation section). If necessary, use treatment to induce expression or modification.

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Problem: Low Signal

The protein of interest cannot be detected after a short exposure of blot to film.

Watch these clips on how to overcome a low signal problem

How to Improve Samples

How to Optimize the Protocol

Lysate Preparation

20-30 µg total protein from whole cell extracts per lane is usually sufficient for detection. If basal levels of target protein, or protein modification are low, it may be necessary to induce expression or modification via chemical stimulant. You may want to investigate alternative cell lines or tissues in which the protein of interest is more abundant. Samples should always be lysed in appropriate buffers that include protease inhibitors and phosphatase inhibitors, for phospho-targets (Phosphatase Inhibitor Cocktail (100X) #5870, Protease/Phosphatase Inhibitor Cocktail (100X) #5872, Protease Cocktail #5871). Lysates should always be sonicated to ensure efficient protein extraction of chromatin and membrane-bound targets. Please visit the Controls Table on our website for suggested positive controls and treatments.

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Primary antibody dilution and/or incubation

Incubate primary antibody overnight at 4°C in TBST at the recommended dilution with the recommended blocking agent (5% BSA or 5% nonfat dry milk). The use of alternate blocking agents, such as gelatin, serum, protein-free blocking agents, casein, or mixed blocking agents may reduce signal. For optimal results with individual antibodies please consult the product datasheet for recommended dilution buffer.

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SDS-PAGE Gel selection

In general, Tris-Glycine gels are recommended, however for high molecular weight proteins we recommend Tris-Acetate gels and associated buffers. Proteins transfer more efficiently from 1 mm gels than from 1.5 mm gels. The use of thicker gels can result in incomplete transfer of high molecular weight proteins so we recommend monitoring transfer efficiency and implementing modifications of transfer conditions to optimize for the size of your protein target of interest.

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Transfer and Membrane

Incomplete transfer can be corrected by longer transfer or by the use of higher voltage. In general, we recommend including 20% methanol in transfer buffer, and performing a wet transfer for 1.5 hr at 70 V. For high molecular weight proteins, we recommend reducing the methanol in transfer buffer to 5% to improve transfer efficiency and avoid fixing large proteins in the gel matrix, as well as increasing transfer time to 3 hr at 70 V. Over transfer (protein transfer through membrane) can be problematic when examining smaller proteins. For small proteins, it is important to use a 0.2 µm pore size membrane and wet transfer for 1.5 hr at 70 V, rather than a 0.45 µm pore size membrane or transferring overnight, which may result in over transfer and a lack of small protein signal.

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Blocking

Blocking the membrane for too long can obscure antigenic epitopes and prevent the antibody from binding. Block for only 1 hr.

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Washing

Washing for longer that the recommended 3 x 5 min is common, and can result in reduced signal. This applies to washing steps after both primary and secondary antibody incubations. Additionally, we recommend washing in TBST, as washing in PBST has been shown to result in reduced signal.

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Secondary antibody dilution and/or incubation

Serial dilutions of the secondary antibody can be performed on blots with the same cell extracts and primary antibody to optimize secondary antibody concentration. Always incubate the secondary antibody in 5% nonfat dry milk in TBST for 1 hr at room temperature. Avoid including sodium azide in HRP-conjugated secondary antibody buffers as azide inhibits HRP activity. CST™ secondary antibodies have already been optimized and do not require titration.

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Detection reagent

Use biotinylated molecular weight standards that can be detected with anti-biotin-HRP as positive controls for chemiluminescent detection.

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