In addition, we’ve received some questions that are frequently asked – hence called frequently asked questions or FAQs – about primary and secondary antibodies when doing chemiluminescent Western blots. So here they are. I am sure there are more. . .so send them our way by commenting on this post!
Why is the signal missing in the middle of the bands?
Too much secondary antibody on the membrane results in consumption of all the substrate in that area. Without substrate, there is no chemiluminescent signal and a white spot appears in the center of the band. Try different dilutions of the primary and secondary antibodies to find what gives the best results, or try changing the substrate.
Does it matter where I purchased the HRP-conjugated secondary antibodies?
The reactivity of secondary antibodies ranges widely between vendors. As well, the ratio of HRP enzyme to antibody varies, and may affect the detection of the target. If the secondary antibodies from one vendor are not working, trying antibodies from other vendors may be helpful.
Should the HRP-conjugated secondary antibodies be highly cross-adsorbed?
Although highly cross-adsorbed antibodies are essential for two-channel, multiplex detection, it is not always necessary with chemiluminescent blotting for a single target.
LI-COR interviewed Dr. Go van Dam, a surgeon specializing in oncology at the Groningen University Medical Center in the Netherlands.
A key focus of van Dam’s research is to explore new tools such as targeted fluorescent imaging probes that will help address the challenges facing oncology surgeons. He discusses his research using near-infrared fluorescent imaging during surgery to improve cancer patient outcomes. Watch this interview with Dr. van Dam.
Vasilis Ntziachristos, PhD, Technische Universität München, Germany and Gooitzen M. van Dam, MD, PhD, University Medical Center Groningen, Netherlands presented “Shining New Light on Clinical Fluorescence Imaging” at World Molecular Congress in San Diego, CA in September 2011.
Okay, so you’ve done your experiments, run your sample on a gel, and transferred the proteins to a membrane. Now, you need to see if you can detect the protein, what happened to it, how much is there, etc.
After you block (remember we talked about how important the right blocker is), you will probe with a primary antibody (that is, an antibody produced to detect a specific antigen) to see your molecule of interest. Now, primary antibodies can be produced in a wide variety of species such as mouse, rabbit, goat, chicken, rat, guinea pig, human, etc. There are lots of suppliers of antibodies out there, so it is important to realize primary antibodies for the same antigen can perform very differently. It may be necessary to test multiple primary antibodies for the best performance in your Western blot system.
In the images below, you can see how different primary antibodies to the same target may react. Serial dilutions of NIH/3T3 lysate were probed with Akt monoclonal primary antibodies from three different vendors. All blots were blocked with 5% skim milk and detected with HRP-conjugated Goat Anti-Mouse and SuperSignal® West Dura chemiluminescent substrate. Western blots were imaged on the Odyssey Fc Chemi channel for 2 minutes, shown with normalized image display settings. You can see that the primary antibodies varied quite a bit. The number and intensity of bands you can detect and the amount of non-specific binding that occurs are definitely different for each one.
So, take a cue from ancient Greece and get to know your Primary Antibody by doing some testing and optimization.
Okay, it’s football season, and I thought the analogy fit. Seriously, the right Blocking Buffer is critical to getting that great chemiluminescent Western blot.
Incubating the membrane in blocking buffer after the transfer step will result in enhanced sensitivity of your blot. Blocking buffer contains proteins that stick to the membrane, promoting specific binding of the primary antibody and minimizing non-specific interactions. Various blocking buffers are available, and it’s important to try several blockers to find the optimal solution for each antigen and antibody pair. There is not a best blocker for all conditions – so you will need to do some testing.
One very, very, very important thing to keep in mind is that the blocker used with HRP-conjugated secondary antibodies in the secondary antibody incubation step of chemiluminescent Western blotting cannot contain sodium azide.
Why?, you ask.
Well, sodium azide binds irreversibly to the HRP enzyme, inhibiting the binding of the substrate and slowing the chemiluminescent reaction. This results in less light production that may affect the appearance of less intense bands or even the entire blot. See the figures below – the blot on the left was done with blocker that did not contain azide; the Western blot on the right used Western blocking buffer with azide.
Nota bene:Odyssey® Blocking Buffer (PBS or TBS) (which does contain sodium azide) CAN be used to block the blot and to dilute the primary antibody but not to block or dilute in the secondary antibody incubation step when using HRP-conjugated secondary antibodies.
On Another Note: Milk is a common blocking buffer; however, milk-based blockers that contain endogenous biotin and glycoproteins may result in higher background on the membrane when detecting with streptavidin. Milk may also contain active phosphatases that can de-phosphorylate phosphoproteins on the membrane.
In the last post, I mentioned that high background on your chemiluminescent Western blot could be due to excess, pooled substrate or letting the blot dry out. So how do you keep the blot moist?
Well, you can place a clear, flat plastic covering on the blot to prevent it from drying out. This will keep the substrate in contact with the HRP enzyme and keep the blot moist. However, there are a few things to keep in mind when using a plastic covering or wrap.
Image the plastic covering by itself first to determine if it scatters light (which would cause high background). You may need to try several types of plastic coverings before finding the best one.
Handle the wrap and the blot with care. Wrapping the blot in plastic wrap may cause unwanted background, especially if it’s folded or handled roughly. When using plastic wrap it is important to avoid wrinkles, as they scatter light (and, again, give you high background).
Try to avoid leaving fingerprints from pressing on the blot (we’ll talk about fingerprints in another post).
In the image, you can that there is unwanted background caused by the plastic wrap.
An alternative to plastic wrap is to use a clear, flat plastic covering, such as a clear sheet protector (from your local office supply store).
IMPORTANT: Be sure to image the sheet or any plastic you use to see if it fluoresces all by itself! After all, this is a creative use of sheet protectors, but not what the manufacturer had in mind so not what they were QC’d for.