Do I need to activate the membrane in methanol?

You do not need to activate the membrane with pure methanol. To activate the membrane it is enough to submerge it in transfer buffer containing >10% EtOH/MeOH for a few minutes. If you want faster activation, submerge in Ethanol 15 seconds followed by equilibration in Transfer Buffer.

Can I use VerdiBlot with my existing Western blot protocol?

Yes, VerdiBlot is compatible with wet and semi-dry methods. For Wet transfer, you can use your normal protocol without changes.For Semi-dry, check our User Guide to see the conditions to maximize your transfer.

Can the membrane be stripped and reprobed?

Yes, VerdiBlot can be stripped and reprobed with different antibodies. You can use most commercials stripping or homemade buffers. Don't heat the membrane more than 35 °C during the stripping procedure. For examples you can follow some of our Application Notes and the User Guide for more details.

Can I use VerdiBlot for both chemiluminescence and fluorescence detection?

Yes, the membrane is optimized to offer very low background in both detection modes.

Trying VerdiBlot for the first time?

Don't worry, in here we will guide you with everything you need to know to get the most out of your Western Blot.

See application Notes

Before you start

Select your target protein (or proteins)

Select your loading controls (we recommend GAPDH and/or Vinculin, but others are acceptable)

Select your membrane (PVDF or Nitrocellulose) for benchmarking, a side-by-side experiment is the best way to see improvements in your protocol.

Plan for dilutions (e.g: 20, 10, 5, 2.5, 1.2, 0.6, 0.3, 0.15, μg). We have detected target protein with loading as little as 0.15 μg of lysate. (this will depend in quality of Ab)

Running the WB

Activate your membrane (no methanol needed)
15 s in EtOH >> 2 min Transfer Buffer >> Ready to GO

Wet Transfer (30-90 V, 60-120 min)
Optimize based on your desired protein MW. For sharp bands always go for low voltages and higher times (Start with 45 V, 2h).

Semi-dry transfer (0.4 A constant, 25 V, 20-30 min)
Follow the recommended settings to ensure good protein transfer without errors. See better results with chilled buffer.

If you are using Turbo conditions (< 10 min) with BioRad Trans-Blot Turbo buffer then please use 0.8 A (constant), 20 V and 7-10 min. See better results with chilled buffer.

See the results

Block the membrane with your usual blocking buffer

Add primary antibody at your usual dilution

Add secondary antibody at your usual dilution

Compare the membranes!
- Qualitative test- Are all the target bands there?
- Quantify the band intensity- Use your favorite imaging software to see the real band intensity
- Background- compare the background between membranes, specially at long exposure times.

Stripping and Reprobing (Optional)

VerdiBlot is suitable for stripping and reprobing. Optimization is always recommended but this guide walks you through the main process.

Simplified protocol
Start with mild conditions. Submerge the membrane in stripping buffer (S. buffer: 0.1 M glycine, pH 2.2, 0.1% SDS, 1% Tween-20) and agitate for 10-20 min. Rinse the membrane with TBST/PBST buffer for 10 min (3X). Before continuing, add ECL substrate and develop the blot, you should see no signal, this indicates the initial Abs were successfully stripped away. If signal is present, repeat step 1. Block the membrane and proceed to reprobe with primary and secondary antibody as you did the first time (different primary Ab). For more details see the Full Guide here.

Compatibility of the membrane

Detection method

Fully compatible with Chemiluminescence and Fluorescence detection.

Fully compatible with monoclonal and polyclonal antibodies

Blocking agents

Fully compatible with common blocking agents:
Casein, dry milk, BSA, synthetic and commercial brands (e.g: Azure Blot Blocking buffers)

Total Proteins Stains

Ponceau S, Coomassie Brilliant Blue, colloidal gold, and more.

See other Results

Frequently asked questions

FAQ EN