JOIN THE AZURE INSIDER CLUB

For the latest publications, promotions, and news on upcoming products sent weekly to your inbox

ASK A SCIENTIST

Got a question? Let us help! Describe the problem you’re having and one of our experts will reach out.

In a previous post I talked about bendy bands, a common problem with improper gel casting and running. However, whilst transferring would seem to be an easier protocol, things can and do go wrong. Below I cover several helpful hints to get you back on track to detecting your proteins after transferring.

No bands after the transfer step: what does it mean?

Unfortunately, the first time you’ll likely consider that your transfer hasn’t worked is when you’re cursing in the darkroom as bands fail to materialize (or in front of your digital imager in the lab, but you’re less likely to be cursing there). That’s probably what led you to this post. 

Here are three steps you can try to eventually detect your proteins.

STEP 1: Simple transfer check

Many of us use a pre-stained ladder in our gels, a simple easy check of transfer efficiency is to check your gel for any residual dye. Your ladder should have transferred across in its entirety, if it hasn’t, something may be wrong in your protocol. If this is the case: try a longer, cooler transfer and remake your buffers.

QUICK TIP: If you see absolutely nothing, check the polarity of your transfer. Run the proteins out of your gel and into the filter paper, rather than the membrane is an easy mistake. But usually one you’ll only make once. Consider color coding the sandwich equipment and leads to ensure the correct polarity at all times.

If you see absolutely nothing, check the polarity of your transfer. Running the proteins out of your gel and into the filter paper, rather than into the membrane is an easy mistake. But usually one you’ll only make once. Consider color coding the sandwich equipment and leads to ensure the correct polarity at all times.

STEP 2: Perform a Detailed Transfer Check using Ponceau Stain

If you want more information about your bands, using Ponceau stain is a great, simple step to validate your transfer. Use Ponceau stain immediately after transferring your membrane. This will be a great way to check for efficient transfer across the length of your lanes.

Comparison of protein transfer using ponceau stain from Azure Biosystems
Azure Ponceau was used to check protein transfer before Western blotting to detect solubilization of a protein of interest from inclusion bodies. Duplicate gels were run with the same set of protein samples and (a) one gel was stained with Coomassie blue. The other gel was transferred to a PVDF membrane, and (b) transferred protein was detected by staining the membrane with Azure Ponceau. (c) After destaining with water, the membrane in (b) was subjected to Western blotting to detect the 72-KDa protein of interest. Lanes 1-3, solubilized inclusion bodies. Lanes 4-6, insoluble pellets. The Western blot was detected using Radiance HRP substrate.

QUICK TIP:  While fine for PVDF and nitrocellulose membranes, Ponceau S will permanently stain nylon membranes. If you see poor transfer across the length of your lane, repeat the above advice: try a longer, cooler transfer, and remake your buffers.

STEP 3: Check Your Protein's Molecular Weight

If your protein has either a high (<120 kDa) or low (>25 kDa) molecular weight then you should consider altering your transfer conditions. For large proteins consider increasing the transfer time, sometimes even overnight, but keeping your transfer cool is key. For smaller proteins consider decreasing the transfer time and reducing the voltage to prevent proteins completely passing through your membrane.

The composition of your transfer buffer can have a huge impact on how readily your proteins move and bind.

For high molecular weight proteins, SDS can promote movement out of the gel and onto the membrane, whereas methanol promotes membrane binding but can inhibit transfer. Consider slightly reducing methanol to between 5-10% and adding SDS to a concentration of 0.1%.

For very small proteins which face the other problem, SDS should be eliminated from the transfer buffer, and methanol concentration increased up towards 20%.

A Ponceau S stained membrane showing efficient protein transfer. Transfer efficacy at higher and lower molecular weights is somewhat reduced.
A Ponceau S stained membrane showing efficient protein transfer. Transfer efficacy at higher and lower molecular weights is somewhat reduced.

QUICK TIP:  For very large proteins, it may be worth considering using a lower percentage gel to allow easier migration of proteins into the membrane. Conversely, small proteins readily leave the gel and pass into the membrane, to prevent transfer through check that your membrane has the smallest pore size of 0.2 μm.

Tried everything, and still no bands?

If at this point you’re transferring like a pro, but still aren’t seeing anything, keep reading. If you’re working with a very low-abundance protein or with a poorly validated antibody, at this point it is worth checking for something that should definitely be expressed. We’re here to help- fill out the form on this page to ask a Western blotting expert a question. Ruling out problems with your probing and developing is the next step; I’ll cover that in a later blog post.

Frequently Asked Questions

Ensure your sample buffer contains all of the ingredients needed, in the right concentrations, and that nothing has expired. If there is an issue with migration, it is probably best to make fresh sample buffer to be safe. Read more

Polyacrylamide gels form a crosslinked, mesh-like matrix through which proteins migrate with the application of current. The matrix functions like a sieve, allowing smaller, low molecular weight proteins to migrate quickly through the gel, while large, high molecular weight proteins tend to progress more slowly. When the electrophoresis is finished, proteins in each lane will be separated into a continuum, with the highest molecular weight proteins remaining near the loading site and the lowest molecular weight proteins reaching the opposite end of the gel. Read 6 tips on troubleshooting band separation

Gel documentation system use light from a wide variety of light sources and filters
to detect fluorescent dyes up to 832nm. contain an ultraviolet (UV) or visible (blue or white) transilluminator, a hood to block the samples from external light sources and a high-resolution camera for image capture.

QUANTITATIVE WESTERN BLOT BASICS
quantitative western blot basics

CLAIM YOUR FREE QUANTITATIVE WESTERN BLOTTING BASICS GUIDE

Get a quick overview of the steps you can take to ensure your Western blots are quantitative. This free guide also includes a troubleshooting section and tear-out quantitative Western blotting checklist.

Let us show you just how easy getting good data can be. Fill out this form to be contacted by a life science expert today!

Shopping cart0
There are no products in the cart!
Continue shopping