Tips and Tricks

Western blotting is a popular protein detection method for studying proteins. Despite its popularity, to obtain reproducible and reliable results is not exactly the norm when performing western blotting. Here, we will provide tips and tricks to help with your western blot optimization including helpful tips on protein sample preparation, blot processing and detection. Request your free Western blot tips, tricks and troubleshooting guide.

General western blotting

Tip 1: Gel electrophoresis

Loading sample in your gel an issue?

Use gel loading tips for a better experience. These tips are thinner, allowing you to insert the tip deeper into the well and not cause overflow.

Want an even better experience? Use Invitrogen precast gels with WedgeWell format. These gels allow you to load up to 60 μL of sample with a typical pipette tip—no need for special tips.

Tip 2: Protein transfer

High molecular weight protein transfers an issue?

Add 0.01–0.05% SDS to the transfer buffer to help pull proteins from the gel onto the membrane.

Want a better way? Use our Invitrogen NuPAGE Tris-Acetate Protein Gels, recommended for better resolution of proteins 150 kDa and above, and our Invitrogen iBlot 2 Gel Transfer Device for efficient 7-minute transfer of high molecular weight proteins.

Tip 3: Blocking

Blotchy blot?

Try filtering your milk blocking buffer for better results. Dry milk powder often does not dissolve completely, and milk solids can interfere with your western detection.

Want to try something easier? Use our Thermo Scientific Pierce Clear Milk Blocking Buffer, which does not contain milk solids that can cause interference.

Tip 4: Primary antibody incubation

Not getting a clean western signal?

If you are using an antiserum instead of a purified primary antibody, this might cause high background. Clean it up using a lysate that does not contain your target protein. Simply add 100 μL of target-free lysate to your antiserum, shake, and incubate overnight at 4°C.

An alternative approach—Use Thermo Scientific SuperSignal Western Blot Enhancer. This not only improves your signal but also decreases nonspecific binding, yielding much better results.

Want to detect multiple targets by chemiluminescence?

Detect 2 or more targets on the same blot by incubating the blot with 2 or more primary antibodies. This will work provided the molecular weights of the targets are different and the gel conditions are optimized to separate the targets from each other.

Want a better experience? Switch to fluorescence mode and use our Invitrogen Alexa Fluor Plus Secondary Antibodies for true multiplex detection.

Reusing your primary antibodies over and over again?

Make sure you add an antimicrobial agent to prevent microbial growth. Sodium azide is not recommended for HRP detection. Use alternative antimicrobial agents such as KATHON™ or ProClin™ preservative at concentrations up to 0.1%. Use your primary antibodies no more than 3 times.

Want a better way? Use the Invitrogen iBind Western System, our automated western blot processing device, for primary antibody savings. For best results, use fresh primary antibody every time. You end up using the same amount of primary antibody.

Tip 5: Wash

Nonspecific bands plaguing your western?

Try using up to 0.05% Tween 20, Triton X-100, or NP-40 detergent in your wash and blocking buffers.

Try an alternative—Use our Thermo Scientific blocking buffers that already have detergent in them.

Tip 6: Secondary antibody incubation

Want to detect PageRuler unstained protein ladders in your chemiluminescence blot?

Just add 1 μL of Strep-Tactin conjugate per 10 mL of your secondary antibody incubation solution. Your Thermo Scientific PageRuler unstained protein ladders (Cat. No. 26614, 26630, 26632 and 26637) can be detected on X-ray film or using imaging equipment.

Want a better way?—Use our Invitrogen iBright Prestained Protein Ladder (Cat. No. LC5615) for both chemiluminescent and fluorescent detection.

Tip 7: Target detection

Detecting peptides on westerns?

Add up to 20% methanol to transfer buffer to boost peptide binding to the membrane. Use a 0.2 μm PVDF membrane to prevent blow through of the peptides.

Want a better way? Use our Invitrogen Novex Tricine Gels for optimum resolution of peptides by gel electrophoresis and our iBlot 2 Gel Transfer Device for efficient transfer of peptides in just 7 minutes.

Not sure if your chemiluminescence substrate is working?

Do a quick darkroom test. Add 1 μL of HRP-conjugated secondary antibody to a working solution of your substrate in the dark. If it glows, your substrate is functional. If not, you need new substrate.

Want a better way? Use our Thermo Scientific SuperSignal West Substrates to obtain fast, reproducible results you can count on.

Having difficulty detecting low abundance proteins?

Increase sensitivity with high sensitivity HRP substrates, or try optimizing current blocking buffer conditions. 5% non-fat milk is a strong blocker and a great choice for high abundant proteins or when there is non-specific binding from your primary antibodies. However, because 5% non-fat milk is a strong blocker, it can over block and suppress signal.

Want a better way? Try SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate, an ultrasensitive enhanced chemiluminescent (ECL) substrate that enables high attogram level protein detection with horseradish peroxidase (HRP) conjugated antibodies. It provides the highest-level sensitivity and better signal-to-noise ratios than other commercially available high-performance HRP substrate with exposures within a fraction of the time.

Try more flexible western blot blockers like StartingBlock Blocking Buffer and SuperBlock Blocking Buffer. These blockers are single purified proteins that provide blocking without compromising sensitivity.

Have limited sample volumes?

Load smaller amounts of your sample by using high sensitivity substrates such as Thermo Scientific SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate. High sensitivity substrates enable you to stretch your samples further and enable loads of 1 µg or less per lane for good signal.

Have limited antibodies?

Use higher sensitivity substrates over standard ECL substrates to get strong signals with lower primary and secondary antibody concentrations.

Want a better way? Get the same detection with smaller amounts of antibodies using SuperSignal West Atto Ultimate Sensitivity Chemiluminescent Substrate. Use primary antibody concentrations from 1:1,000–1:20,000 and secondary antibody concentrations from 1:100,000- 1:250,000.

High abundant targets–go down to 20x less primary antibody (1:20,000)

Low abundant targets–go down to 5x less primary antibody (1:5,000)

Tip 8: Stripping a blot

Stripping blots without getting desired results?

Always wet the blot with wash buffer for 5–10 minutes before adding stripping buffer. Use homemade stripping buffer and heat the blot at 37°C for up to 30 minutes.

Want a better way? Try our Thermo Scientific Restore PLUS Western Blot Stripping Buffer for efficient stripping in just 10 minutes at room temperature.

Fluorescent western blotting

Tip 1: Gel electrophoresis

Want to avoid high background?

Sample buffers containing bromophenol blue will fluoresce and can contribute to increased background. If using sample buffers with bromophenol blue, the dye front may be run off the gel prior to transfer or cut from the membrane after transfer to avoid background fluorescence signal.

Want a better way? Consider using fluorescence-compatible sample buffers without bromophenol blue, such as Invitrogen Fluorescent Compatible Sample Buffer.

Want to decrease background fluorescence and signal bleed-through to adjacent lanes?

Decrease the amount of molecular weight markers loaded onto the gel. Use single-color, prestained molecular weight markers, as multicolor markers can appear in distinct channels.

Want a better way? Use protein ladders specifically designed for western blotting, such as Invitrogen iBright Prestained Protein Ladder, which provide prestained proteins as well as fluorescent bands for detection. Typically, 2–4 μL is sufficient.

Tip 2: Protein transfer

High background due to incorrect choice of membrane?

The nature of the membrane can affect the background. Choose membranes with low autofluorescence, including nitrocellulose and specialty low-fluorescence PVDF membranes. Also, avoid using pens on membranes, as many inks fluoresce; use a pencil instead.

Want a better way? Use Invitrogen Nitrocellulose/Filter Paper Sandwich and Thermo Scientific Low-Fluorescence PVDF Transfer Membrane to overcome this issue.

Tip 3: Blocking

Is membrane contamination (speckles and fingerprints on the membrane) an issue?

Handle membranes with gloved hands and clean blunt forceps. Also, use only high-quality filtered blocking buffer to help prevent particles and contaminants from settling on membranes.

Want a better way? Consider using Thermo Scientific Blocker FL Fluorescent Blocking Buffer, which is designed to reduce cross-reactivity and produce high signal-to-noise ratios. Do not add detergent to blocking buffer, as this may increase background fluorescence.

Fluorescence detection a pain with mammalian samples?

Use a nonmammalian blocker like salmon serum to maintain low backgrounds and high signal-to-noise ratios.

Want a better way? Try our Thermo Scientific Fish Serum Blocking Buffer for fluorescence detection. Made from steelhead salmon serum, Fish Serum Blocking Buffer is especially useful as a blocking agent in fluorescence detection methods involving mammalian samples.

Tip 4: Imaging

Don’t see signal or have weak signal in blot?

Increase exposure time to obtain a better signal.

Want a better way? Utilize the Smart Exposure feature to obtain an optimal image on the Invitrogen iBright FL Imaging System.

Tip 5: Stripping a blot

Stripping blots without getting desired results?

Traditional stripping buffers may only be effective for removing low-affinity antibodies, leaving behind dye-labeled secondary antibodies that can overwhelm the target signal when reprobing. Choose a stripping buffer that is specific for use in fluorescent western blotting.

Want a better way? Try our Thermo Scientific Restore Fluorescent Western Blot Stripping Buffer, which is gentle and designed for quickly removing primary and near-infrared (IR) dye–labeled secondary antibodies from western blots. This stripping buffer is for use with low-fluorescence PVDF membranes only.

For Research Use Only. Not for use in diagnostic procedures.