Western Blot Troubleshooting

 

ProSci Inc has answers for all your western blot troubleshooting needs. Consult our western blot protocol and these valuable western blot tips for a variety of issues and problems.

Challenge: Western blot signal is weak or undetectable

Inadequate Protein Binding

For western blot troubleshooting weak signal, simply increasing the concentration of antibody and/or extending the incubation time at the appropriate temperature can support adequate binding to the protein of interest by the primary or secondary antibody. In the case of inadequate antigen in a given sample, it is recommended that a minimum of 15 µg of protein be loaded in each lane of the gel, and for recombinant protein < 1ng. It is also important to incorporate protease inhibitors into the sample.

Antibody Incompatibility

When experiments call for procedures involving primary and secondary antibodies, it is not uncommon to experience incompatibility. One method of troubleshooting Western blot challenges of this kind is by maintaining a consistent species in both antibodies. For example, an anti-mouse secondary antibody should be used with a primary antibody raised in a mouse.

Challenges with Blocking Agents

The high protein affinity of membranes commonly used for Western blotting procedures promotes retention and binding of transferred proteins. Incubating the membrane with a blocking agent to prevent nonspecific antibody binding helps ensure proper detection of transferred proteins. It is important to avoid excessive washing of the membrane. In the event of a Western blot problem such as a cross-reaction, introducing a mild detergent such as Tween 20 can help alleviate any interference between the antibodies (primary or secondary) and the blocking agent. For determining whether non-specific binding is caused by the secondary antibody, try running a secondary control in the absence of the primary antibody. Proper optimization of blocking agents can help significantly reduce background noise.

Protein Transfer Challenges

A good first step is to determine whether or not the transfer was performed in the correct direction. A reversible stain can be particularly useful in this regard. Whether using a nitrocellulose, nylon or PVDF (polyvinylidene difluoride) membrane, it is important to ensure that the membrane is properly prepared. In the case of PVDF, a methanol pre-treatment is required prior to introducing transfer buffer. It is important to note that each membrane type presents different advantages. For example, the PVDF membrane is associated with a lower background in comparison to the nitrocellulose membrane.

Challenge: Optimization of Signal-to-Noise Ratio

High Concentration of Primary Antibody

To help maintain a balanced concentration of primary antibody in the sample, it is often effective to extend the incubation period but at a more diluted concentration. Ideally, this should be performed in a steady fashion overtime. However, during the incubation process, necessary precaution should be taken to prevent drying out of the membrane. Similarly, additional and more frequent washes may be required to adequately remove unbound antibodies.

Challenge: Bands, Spots and Dots

Appearance of White or Black Spots on blot

A common reason for the appearance of uneven white spots is trapped air bubbles. It is important to take special care to remove any bubbles during gel preparation. Similarly, filtration of the blocking agent can help prevent the formation of black dots often caused by the binding of antibodies to the blocking agent.

Undesirable Bands:

Bands too close together. Insufficient separation of bands can be remedied by adjusting the gel percentage. In this manner, smaller proteins should be run in a higher gel percentage and the opposite gel percentage for proteins larger in size. In the event of an excessively fast migration, a decrease in the voltage level can help balance speed. Alternatively, if a migration proceeded at an excessively warm temperature, running the gel on ice is an effective method of western blotting troubleshooting.

Multiple bands

A higher dilution of either the primary or secondary antibody can help eliminate unrelated, weaker bands when troubleshooting western blot multiple bands. Extra bands may also be caused by an excess amount of lysate loaded onto the gel. Try increasing the wash cycle may help.

Non-specific bands

Non-specific bands could be caused by the secondary antibody alone. Try running a western blot with and without a primary antibody then probe with the secondary antibody and compare the signals. For more information on western blot troubleshooting please contact techsupport@prosci-inc.com or give us a call.