The best transfer conditions, membrane, and blocker for experiments depend on your antigens and antibodies. For data examples and troubleshooting tips, see the Good Westerns Gone Bad technical note.
Always handle membranes carefully and with clean forceps. Before you use them, clean dishes, bags, or trays for incubations with methanol.
Different primary antibodies will react differently in different blocking buffers. Optimize to determine the best blocking buffer for your primary antibody
Don’t use milk for blocking, as milk typically contains IgGs that cross-react with anti-goat secondary antibodies. Instead, try an Intercept® Blocking Buffer.
Add Tween® 20 to primary, secondary, and wash steps at 0.1 – 0.2%.
For PVDF membranes, add SDS to secondary antibody incubation (not primary antibody incubation or wash step) at 0.01 – 0.02%.
Do not add detergent to the blocking step.
With IRDye® 680LT Secondary Antibodies, use SDS (0.01 – 0.02% final concentration) and Tween 20 (0.1 – 0.2% final concentration) during the detection incubation step.
Wash 4 times, for 5 minutes each wash. If background persists, increase the number of washes and buffer volume. Make sure that 0.1 – 0.2% Tween 20 is present in buffer. Excess Tween 20 (0.5 – 1%) may decrease signal.
Do not add secondary antibodies at concentrations higher than 1:5,000, as high background will result. For IRDye Secondary Antibodies, a good starting dilution is 1:20,000. Use the MPX™ Blotting System to optimize primary and secondary antibody dilutions. Get the One-Blot Western Optimization: Using the MPX Blotting System technical note for more information.
IRDye Secondary Antibodies are stable for 3 months at 4° C. After 3 months, the effectiveness of secondary antibodies will decrease, and background may increase.
Increase antibody volume so entire membrane surface is sufficiently covered with liquid at all times. Use enough reagents to prevent areas of the membrane from drying out during incubations and washes. Gently agitate during every antibody incubation.
Use low-fluorescence PVDF membrane, and confirm uniform membrane background before you transfer. LI-COR prescreens Immobilon-FL PVDF membrane kits for quality control.
Blocking solutions containing BSA may cause high membrane background and nonspecific antibody binding for near-infrared Western blots. Avoid BSA for blocking.
Your data will not be quantitative if you incubate multiple membranes together in one container. If you must block multiple membranes in the same dish, use enough blocking buffer for all membranes to move freely and fully contact the blocker.
If using PVDF, pre-wet the membrane in 100% methanol until it becomes translucent gray instead of opaque white. Wet membrane in PBS or TBS for 5 minutes or until uniform in color. Then place the membrane in blocking. Pre-wetting is necessary to help the PBS or TBS interact with the membrane, because PVDF membranes are very hydrophobic.
If using nitrocellulose, wet membrane in PBS or TBS for 5 minutes, or until uniform in color, before blocking.
After blocking, keep membrane completely wet at all times during the blotting process. This is especially important if you’re planning to strip and reprobe the blot.
Avoid touching membranes with gloved or ungloved hands, as fingerprints will fluoresce on the Odyssey® Imaging System. Always handle membranes with clean forceps, free of any contaminants or antibody solutions. Clean forceps with methanol before using or after dipping them into an antibody solution (especially dye-labeled secondary antibody). Dirty forceps deposit dye on the membrane that you can’t wash away.
Clean dishes, boxes, or trays with methanol before using them for incubation.
Clean scanning surface and mat carefully before each use. Dust, lint, and residue will cause speckles.
Use pencil to mark membranes. For nitrocellulose membranes, you can also use an Odyssey Pen.
Transfer pads in wet tank systems and transfer boxes accumulate residue after frequent use that can cause speckles on Western blot membranes. Clean transfer pads and transfer boxes by soaking them in 100% methanol for 10 minutes.
Always clean your imaging system before you image. For the Odyssey CLx, and Odyssey Classic Imaging Systems, clean the glass surface with an alcohol-based cleaner before imaging.
For the Odyssey Fc Imaging System, avoid using imaging trays that have been used for Coomassie stained gel imaging. Clean trays with an alcohol-based cleaner before you image.
Primary antibody may have low affinity for your target. Increase amount of antibody or try a different supplier.
Extend primary antibody incubation time. Try 4-8 hours at room temperature, or overnight at 4 °C. Do not reuse antibody.
Increase amount of primary or secondary antibody, optimizing for best performance. Try the MPX Blotting System to optimize antibody concentration. For details, get the One-Blot Western Optimization: Using the MPX Blotting System technical note.
Primary and secondary antibodies can lose reactivity from improper or extended storage. Examine the product’s shelf life, and consider replacing with fresh antibody stocks. Avoid reusing antibody.
Decrease Tween 20 in diluted antibodies. Recommended Tween 20 concentration is 0.1 – 0.2%.
For PVDF membranes, recommended SDS concentration is 0.01 – 0.02% during the secondary antibody incubation step (in addition to Tween 20). Some antibodies require an even lower concentration.
For nitrocellulose membranes, do not add SDS to any steps.
Your primary antibody may work much better with a different blocking buffer. For tips on how to choose an appropriate blocker, get the Western Blot Blocker Optimization for Near-Infrared Detection protocol.
Loading too little or loading too much protein sample will decrease antibody sensitivity. Loading too little sample can result in not enough antigen present. Loading too much sample can result in your target antigen being masked by other proteins or antibody hindrance. Determine the optimal loading concentration by performing a serial dilution of your target of interest.
Try loading a dilution series that ends with the original amount of antigen that didn’t produce enough signal. This helps you determine the best signal with the lowest amount of antigen. You can also use the narrowest possible well size to concentrate antigen.
Check transfer buffer choice and blotting procedure. For more details, get the Protein Electrotransfer Methods technical note.
Wet tank transfer is the gold standard for protein transfer. Validate your transfer method to ensure your target antigen transfers under the preset conditions. The amount of methanol in the transfer buffer, timing of gel presoak, choice of membrane, voltage, and length of transfer can all change how much protein transfers to the membrane.
A pre-stained molecular weight marker can help you monitor transfer. You can also use REVERT™ Total Protein Stain to stain membranes post-transfer to monitor transfer efficiency. Stain gels with Coomassie blue after transfer to see if the gel retained any proteins.
After transfer is complete, dry your membrane before you block. Drying the membrane allows proteins to bind tightly to the membrane, preventing potential signal loss.
For PVDF membranes, re-activate membranes with methanol and rinse with water before blocking.
SDS in transfer buffer may interfere with binding of transferred proteins, especially for low molecular weight proteins. Try reducing or eliminating SDS. Note: presence of up to 0.05% SDS does improve transfer efficiency of some proteins.
Air-dry membranes completely for 1 hour (or 10 minutes at 37 °C) after transfer, to make binding irreversible.
Transfer of large proteins (>140 kDa) often requires lower methanol concentrations (10%) and possibly the addition of SDS to 0.05%. To get complete transfer of larger proteins, extend transfer times.
Small proteins may pass through the membrane during transfer (“blow-through”). To get good membrane retention of these smaller proteins, use higher methanol concentrations (30 – 40%) and lower voltage transfer.
Extended blocking times can mask antigen and decrease signal intensities. Avoid blocking membranes for more than 1 hour. Block membranes at room temperature. For the best experimental replicability, block membranes at consistent times and temperatures.
The blocker you use may affect background bands. If you encounter high background or unexpected bands, try a different blocker. For tips on how to choose an appropriate blocker, get the Western Blot Blocker Optimization for Near-Infrared Detection protocol.
In two-color Western blots, antibody cross-reactivity is always a possibility. To avoid cross-reactivity:
Check your fluorescent dye. Fluorophores like Alexa Fluor® 750 may appear in both channels (700 nm and 800 nm) and are not recommended for use with Odyssey Imaging Systems.
If signal in one channel is very strong (near or at saturation), it may cause signal in one channel to bleed to the other channel. Minimize this by using a lower scan intensity setting in the channel that had strong signal. If you have an Odyssey CLx Imaging System, try the AutoScan mode.
Reduce signal by reducing the amount of protein loaded or the amount of antibody. A dilution series of each will also help you save on sample and antibody.