How to Western Blot

Densitometry Tutorial for Image Studio Lite from LI-COR

administrator — Wed, 27 Feb 2013 16:21:08 +0000

LI-COR Biosciences has launched a new piece of free software for the analysis of western blot gels and film called Image Studio Lite. I have provided a tutorial for the use of this new software for simple densitometry analysis.

Image Studio Lite Tutorial

Please check out the new tutorial. I think the new software has some definite advantages over traditional ImageJ methods. In particular, Image Studio Lite allows you to quantify bands in a single click, whereas ImageJ requires a multi-step process. It will probably be a matter or personal preference between these two free programs, but it is definitely at least worth a look at Image Studio Lite to see how you like it.

Comments and Help

administrator — Fri, 19 Oct 2012 15:11:11 +0000

After some time away, I am back and ready to address some questions regarding western blot quantification. I hope you’ll all excuse the delay, but real life and research have been a bit overwhelming the past month. I will try to reply to all of the backed-up comments as quickly as possible.

Please feel free to help each other in the discussion, as many of you seem to have had similar issues with ImageJ software, and, more often than not, there is usually a quick-fix solution.

Also, due to the vast amount of spam I was receiving, I needed to change some commenting policies. Now, you will have to be signed-in to your account to comment. Hopefully, this policy will result in more efficient Q&A for everyone.

All the best

Free help for new students!

administrator — Sun, 02 Sep 2012 22:24:22 +0000

As new students start to arrive in your labs for the new school year, don’t forget to direct them to our site. We have free and proven protocols for western blotting. We also include troubleshooting, densitometry tutorials, and recipes for common buffers.

Don’t lose out on productivity by letting inexperienced undergraduate and graduate students standardize western blots from scratch. By following our protocol, you will all save time and money.

Good luck at the bench!

Protocol section is expanding…

administrator — Sun, 20 May 2012 16:43:28 +0000

In recent weeks, we’ve seen a large growth of traffic to this site, along with an increase in requests for various protocols and buffers. Because of this, we are expanding our protcol sections to include some basic protein techniques for molecular biology (e.g. immunoprecipitation, gel staining, etc.). Immunoblotting will still be our focus, but we recognize that many other techniques are closely associated with western blots, and we are happy to share any advice for those techniques. If you have any direct requests or questions, contact us directly or post it to the forum.

New Discussion Forum!

administrator — Thu, 17 May 2012 03:17:50 +0000

We have just finished adding a new discussion forum for our site. We sincerely hope that this forum will help the researchers who use our site troubleshoot and share information effectively.

I hesitated to start a forum so quickly, but keep in mind that although this is a relatively new site, we still get thousands upon thousands of unique visitors every month. Please don’t hesitate to post questions or problems in the forum. If I can’t personally answer it, I’m sure another visitor can.

We are in a sort of testing phase. So, we’ll see how smoothly things go with this forum for now. Please let us know if you notice any serious issues or bugs. We hope the new forum is helpful!

Western Blot Overview Page is Up!

administrator — Thu, 10 May 2012 02:36:35 +0000

After a long wait, we have finally posted our general western blot overview. This may be useful for beginners who are just learning the basics of western blotting technique, or people outside of the field who want to understand how western blotting works. There are some good figures of how proteins are resolved via electrophoresis, and all of the basic steps are covered.

Western Blot Stripping Buffer Recipe and Protocol

administrator — Sat, 05 May 2012 18:17:59 +0000

Western Blot Stripping Buffer:

Making your own western blot stripping buffer is easy and affordable. We essentially always use PVDF membranes for our western blots because of their durability and low background for a variety of primary antibodies. PVDF membranes are useful because they can be stripped and re-probed many times to analyze multiple protein targets on the same membrane. This is a time and money saver because it allows us to look at multiple targets, without having to run multiple gels on the same samples. It is also a more technically sound practice to look at various proteins within one blot, rather than comparing between multiple blots (I’m talking about a single experiment. Of course, you’ll want to run new gels for biological replicates).

The recipe for the western blot stripping buffer we use is:

60 mM Tris-HCl pH 6.8
.7% β-mercaptoethanol
2% SDS

To make 500 ml of western blot stripping buffer:

30 ml of Tris-HCl pH 6.8
3.5 ml of β-mercaptoethanol
50 ml of 20% SDS
Add 416.5 ml of ultrapure water.

Western Blot Stripping Protocol:

After developing the previous blot, remove the membrane from the cassette and place it in TBST to remove ECL.
Pour a small amount of western blot stripping buffer into a closed container (under hood because of β-mercaptoethanol). Usually, 5-10 ml is sufficient depending on the size of the container.
Place the western blot membrane into the stripping buffer and incubate in a water bath at 50°C for 30 minutes under gentle agitation.
Remove the PVDF membrane from the western stripping solution and place it into TBST. Be sure to dispose of the stripping solution properly because it contains β-mercaptoethanol.
Wash the membrane 5 times in TBST under gentle agitation to remove all traces of the western blot stripping buffer.
Proceed to the blocking step of the western blot protocol.

Saving Western Blot Antibodies for Reuse

administrator — Tue, 10 Apr 2012 14:04:50 +0000

If you are not currently, reusing primary antibodies for your western blots, you’re washing a lot of money down the drain.

In our lab, we regularly reuse primary antibodies at least 3 times before we get rid of them. Some antibodies will work longer than others. In particular, antibodies for post-translational modifications tend to have a shorter shelf-life in terms of saving and reusing. Nevertheless, storing and reusing primary antibodies is a great way to save your lab some money, since the primary antibody is usually the most expensive part of a western.

We save antibodies by first making a 5% Sodium Azide solution in water (THIS SOLUTION IS TOXIC. HANDLE UNDER A FUME HOOD). Add this solution at a 1/100 dilution to your primary antibody solution (usually in 5% BSA in TBST). This gives you a final concentration of .05% sodium azide. This solution will be good for several weeks at 4 degrees celsius.

Snap i.d. Western Blotting Protocol

administrator — Sun, 08 Apr 2012 19:11:22 +0000

I previously shared a review of the Snap i.d. rapid western blotting system from Millipore. I stated that the product definitely works with a good set of antibodies, but individual researchers must decide for themselves whether the saved time was worth the Snap i.d. price tag.

As a follow-up, I’ll share the protocol that I was using to perform westerns blots using the Snap i.d. apparatus. That way, those of you who do make the purchase will have a better jumping off point for standardizing your protocol. Keep in mind, also, that the Millipore will supply you with a pretty good protocol to use. I adjusted it in a couple places just to optimize reduced background and signal to noise for our lab.

Millipore Snap i.d. Western Blotting System Protocol:

Follow your normal western blot protocol or bis-tris protocol all the way through the transfer step.
After transfer, place the membranes in a tray of TBST to wash the residual transfer buffer away.
Use milliQ water to wet the blot holder. The holder will go from white to slightly translucent as it becomes wet.
Place the western blot membrane into the blot holder with the protein side facing the blot holder membrane.
Snap the blot holder shut and insert the blot holder into the Snap i.d. apparatus. The blot holder well should be facing up now, and the protein side of the membrane should be facing the well of the blot holder.
Being very careful to not let the membrane dry out, turn on the vacuum and pour in 25 ml of 2.5% BSA in TBST into the blot holder well. (The Millipore protocol says to use no higher than 1% BSA, but I found that 2.5% works fine with the machine, and reduces background compared with 1%).
Allow the blocking solution to completely drain from the Snap i.d. well. Once it is drained, turn off the vacuum. If you wish to save your primary antibody solution, insert a recovery dish now.
Add 10 ml of primary antibody solution (in 2.5% BSA in TBST) to the Snap i.d. well and let it incubate for 15 minutes. (Millipore instructs you to use lower volumes, but we found that very low volumes had a tendency to pool in the well of the blot holder, thus resulting in uneven coverage of the western blot membrane.)
After ten minute incubation, turn the vacuum on. Your primary antibody solution will be vacuumed out of the well and, if you have a recovery dish, saved for later use.
Leaving the vacuum on, wash the membrane three times with 25 ml of TBST. Be careful to pout the TBST into the well gently on the sides.
After washing, turn off the vacuum and add 10 ml secondary antibody solution in 2.5% BSA in TBST. Let this incubate for 15 minutes.
After incubation, wash the membrane 4x with 25 ml of TBST. Be sure to let each wash completely drain from the blot holder well before adding the next wash.
Remove the western blot membranes from the blot holders and place into a fresh tray of TBST.
Perform western blot development as normal.

This is a pretty straightforward protocol, and I tried to indicate where it differed from the standard protocol issued by Millipore. Hopefully it will save time for those of you who decide to use this apparatus.

As a final note, we did attempt to reuse blot holders, even though Millipre clearly stated that they are one-use only. As it happens, I must agree with them. After more than one use they tend to accumulate salts which leave clear marks on your membrane and create a strong background signal during development.

Make Bis Tris Gels in your own lab!

administrator — Thu, 29 Mar 2012 03:53:30 +0000

The newest and best polyacrylamide gels for western blotting use bis-tris as a buffer. These gels are sold by Invitrogen under the brand name NuPAGE®. However, these gels can be expensive, and, of course, have a limited shelf life. So it’s really easier if you just make them yourself in your lab.

On this site, we now have completed a full bis tris western blotting protocol for you to use in your own lab. All of the bis tris gel recipes are available in you polyacrylamide gel section. All of this information is available to your for free.

Our lab has completely converted from tris-hcl gels to bis-tris gels, and our results have never been better. If you want to save time and money standardizing the bis tris western blot method, then use our protocol and gel recipes as a starting point.

I hope you find the information helpful. As always, feel free to contact us with any comments, questions, or concerns.