Gould Jannik

Electrophoresis encompasses a wide range of techniques in which charged biomolecules in a liquid, a solid, or a semisolid solution are separated by size by the application of an electric field. The most common application of electrophoresis in biology is the separation of proteins by SDS-PAGE.

Irrespective of how you obtained your protein sample and what its end use is, you need to mix it with a solution to make it compatible with SDS-PAGE. This solution is called Laemmli buffer and is invariant in nearly all protocols.

In this article, we’ll cover the components of Laemmli buffer, what they actually do, and end with a handy buffer recipe for your lab notebook.

Laemmli buffer takes its name from Professor Ulrich K. Laemmli, who refined the SDS-PAGE procedure in the 1970s.  It creates the physicochemical conditions necessary for the high-quality separation of protein analytes based on their molecular weight.

It does so through the requisite blend of the five following reagents:

A smart scientist knows what the reagents in their experiment do, so let’s delve into the chemistry and purpose of the Laemmli buffer components.

Why do we need it?

SDS is a well-known detergent that’s often used to denature proteins. It does so by disrupting non-covalent bonds to destabilize the secondary, tertiary, and quaternary structural assemblies. Denaturing the protein analytes in SDS-PAGE is crucial so that their separation is solely a function of molecular weight.

It is also used to impart a net negative charge onto all of the protein analytes so that they all migrate in the same direction during an SDS-PAGE experiment.

How does it work?

The negatively charged sulfate moieties on an SDS molecule make it an anionic surfactant. The net negative charge it imparts when it binds to protein analytes causes repulsion between amino acids. Including SDS in Laemmli buffer leads to protein denaturation to give a linearized version of the protein analytes.

Since all of the protein analyte molecules will be coated in SDS, the experimenters (you and I) can control their charge by adjusting the pH of the buffer to which they are exposed. By preparing the SDS-PAGE running buffer at pH 8.3 we are effectively ensuring that all of the sulfate moieties are deprotonated (negatively charged). Thus all of the analytes will migrate toward the anode when we turn the power pack on.

It’s estimated that 1.4 grams of SDS can bind 1 gram of protein. 

Why do we need it?

Remember when I said that SDS will break apart analyte quaternary structure? I was lying.

Some quaternary structures are held together by disulfide bonds, which are covalent. Because they are covalent, SDS won’t touch them and a specific reagent is needed to break them apart. Enter the reducing agent.

How does it work?

The common reducing agents beta-mercaptoethanol (BME) and dithiothreitol (DTT) contain thiol (-SH) groups. Without breaking out the mechanisms, the proton in the thiol group is labile.

The deprotonated form of a thiol group is called a thiolate group and it’s negatively charged because it has an extra electron.

This electron attacks one of the sulfur atoms in the disulfide bond and, well, what happens next depends on what reducing agent you are using.

But skip forwards to the end and the reducing agent now contains the disulfide bond (it’s been oxidized) and the sulfur atoms on the protein analyte(s) that were once involved in the disulfide bond are now each bonded to a proton instead (they’ve been reduced to two thiol groups).

Why do we need it?

Tris functions to maintain a pH of 6.8 to keep the Laemmli buffer chemically stable. Tris also inhibits a number of enzymes and so can prevent proteases from degrading your analytes.

Preparing the Laemmli buffer at pH 6.8 also helps achieve maximum resolution for your SDS-PAGE Experiment.

How does it work?

Tris is the conjugate base of the buffer solution. Like all buffer solutions, it can sequester excess H+ or OH– species that may evolve over time due to (say) the degradation of other components of the solution or absorption of atmospheric CO2.

As to why a pH of 6.8 helps achieve maximum resolution– it’s a little complicated.

Basically, pH 6.8 matches that of the stacking gel. pH 6.8 is also close to the isoelectric point (pI) of glycine (6.08). And the SDS-PAGE running buffer uses a tris–glycine (not tris–HCl) buffer system.

In an SDS-PAGE experiment, the tris–HCl, protein analytes, and glycine (from the running buffer) all enter the stacking gel at the same time. The Cl– anions will fly through the stacking gel because they’re relatively small and fully negatively charged.

Glycine on the other hand migrates relatively slowly through the stacking gel because it’s only deprotonated a fraction of the time (the pH of the stacking gel is only slightly above its pI).

The protein analytes, meanwhile, migrate at a speed that’s between the two extremes of Cl– and glycine. This results in a sandwiching effect, and all of the analytes get bunched together prior to entering the resolving gel. This is conducive to sharp, resolved bands when you stain the gel.

Why do we need it?

It’s all well and good doing fancy biochemical alterations to your analytes, but remember that your SDS-PAGE gel is submerged in running buffer. Pipetting in your analytes at this stage would see them just diffuse into the gel tank. Sad times. A good dollop of glycerol can prevent this.

How does it work?

Glycerol is denser than water. Simple.

Mix enough of it into your Laemmli buffer–sample mixture and the whole solution will be denser than water. Thus it will sink when you (carefully) load it onto the SDS-PAGE gel.

The density of glycerol is 1.26 g/cm³.

Pro tip! Glycerol is a difficult liquid to pipette accurately and is much easier to measure out by mass. Simply multiply your desired volume of glycerol by 1.26 to get the mass you need to add and pipette it into your Eppendorf tube on a balance.

Why do we need it?

A dye, usually bromophenol blue is added to the sample buffer and enables us to see our samples as we load them onto the SDS-PAGE gel. It’s not strictly necessary but you need good eyesight to forgo it.

Similar to the Cl– mentioned already, the dye molecules will migrate through the resolving gel much quicker than your protein analytes. All the dye molecules actually migrate together in a line called the “dye front.”

As long as the dye front is still on the gel, you can be confident that even the lightest of your analytes is also still on the gel.

How does it work?

It turns the Laemmli buffer blue (or any color you want).

Here’s something to print out and stick into your lab notebook: an easy table of Laemmli buffer recipes!

A concentrated Laemmli buffer can be stored at 4 °C for at least a year without worrying about its effectiveness. If you don’t have BME you can use DTT instead, but re-add it every now and then because it’s less stable than BME.

So now you have the know-how to prepare your own Laemmli buffer for SDS-PAGE and, better yet, how it works! Have you found this article helpful? Let us know in the comments section below if so.

Originally published November 2019. Reviewed and updated November 2021.

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Historically, wedding speeches date back to ancient societies, when those in attendance would raise their glasses to their gods. Today, giving a speech (or toast) to honor the newlyweds is still a big part of wedding celebrations.

Typically, those expected to give wedding speeches usually includes the newlyweds' parents, one representative from each side of the couple's wedding party (the best man and maid of honor), and a response toast from the newlyweds, explains Allyson Joseph, an event producer at Bob Gail Events in Los Angeles, California.

As for rehearsal dinner speeches, it's most typical for the father and mother of the groom to raise a toast since they often host this event — while the wedding is commonly hosted by the bride's family.

But there are no set-in-stone rules about who should (and shouldn't) give wedding speeches. It really depends on the occasion, as well as the couple's preferences and culture. Here, several wedding experts provide their very best wedding speech tips.

You've heard of "wedding speeches" and "wedding toasts" — but what's the difference? Here's what experts have to say.

"A toast is generally an acknowledgment of a timely event, in the case of a wedding toast, to honor and congratulate the happy couple on their nuptials," says Diane Gottsman, an international etiquette expert and founder of the Protocol School of Texas.

On the other hand, Gottsman says "a speech is a 'talk,' a lengthier conversation of best wishes and reminiscent of the bride and groom's first meeting, history, or a warm and friendly story."

Julie Blais Comeau, Chief Etiquette Officer at Etiquette Julie, agrees. She says that a "toast is a proposal to drink, to 'cheer,' to honor [the couple's] union and to congratulate them. It is followed by a raising of the glass, the collective taking of a sip to wish them well. It is short and to the point."Speeches are typically longer, and "may include an anecdote or multiple stories and last more than three minutes," says Anne Chertoff, chief operating officer at Beaumont Etiquette.

In sum, you'd raise a toast to the newlyweds and invite everyone to take a sip of whatever they're drinking (often champagne), and speeches are typically pre-written, lengthier tributes.

The time has come to write a great wedding speech. You go to put your pen to paper — only to realize you are totally unsure of what's most important to say. Here, you'll get advice on how to deliver a great speech that holds meaning, plus tips on what not to say.

If it's your first time giving a wedding speech, don't worry if you're not an experienced speechwriter. We recommend starting from the top. What should you begin by saying?

"When drafting a wedding speech, the speaker would start it based on what their role is in the wedding," Chertoff says. For example, "the father or mother of the bride or groom may start by thanking everyone for attending, since they may be the host of the occasion."

If you're giving a maid of honor speech or best man speech, you may want to start by introducing yourself, Chertoff explains. This introduction may include how you know the bride or groom — and if appropriate, you can also throw in some heartfelt memories you have about meeting their new spouse.

This advice — to start by introducing yourself — can apply to any speech by any guest. It's a smooth way to kick off your tribute, which will also provide context for those listening.

The ending of your speech will likely wrap up in a natural way as you write. But, to make sure you hit the right note, consider the following."The end of a wedding speech should summarize the feeling of the speech and the occasion," Chertoff says. "The speaker may want to ask everyone to raise a glass to toast the couple — or they may want to end by saying how much they love the newlyweds."

It's really that simple. Just make sure that your speech has a wrap-up of some kind, end it on a heartfelt note, and don't just stop abruptly after telling a story.

When all else fails, you can signal the end of your speech by raising a toast. (After all, everyone loves an energetic toast, followed by a sip of their favorite drink.)

What you say in your wedding speech really depends on your relationship with the couple — and also the type and formality of the affair.You may, for example, want to be extra careful about how and when you "throw shade" at the couple. "While some people may think it's funny to roast a bride or groom, a rehearsal dinner is the more appropriate place for a roast-style speech," Chertoff says.

Along the same lines, you may want to avoid bringing up certain topics. "Steer clear from mentioning past love interests, private jokes or one-liners no one else will understand, or personal details that could potentially be offensive," Gottsman says.

She adds: "Humor is dangerous — unless you are really good at pulling it off and very few people are as funny as they think!"

Finally, it's safest to stay away from curse words — and definitely don't belittle, ridicule, or shame anyone, Chertoff explains. "Remember that what you say and do can be recorded at any moment and could go viral."

With all of your loved ones attending your wedding, you might be wondering who should give wedding speeches on your big day."The list can be lengthy and include everyone in the wedding party, including bridesmaids and groomsmen, if he or she wants to," Blais Comeau says. "There is no obligation on anyone's part."

Blais Comeau adds: "Top of the list is the bride and groom, followed by the father of the bride speech and the father of the groom speech, unto the best man, the maid of honor. Start at the top, decide, ask, and plan."

What's the usual order of wedding speeches? "The best man and the maid of honor traditionally offer the first speech, generally before the meal is served," Gottsman says. Then, "a parent of the couple may make a dessert speech honoring the couple, as well."

If the newlyweds also want to give a speech, that's welcomed. After all, they're the reason everyone is gathered. It's always nice to thank everyone for coming — and show appreciation for all who may have traveled from afar.

Wondering how long your wedding speech should be? Here's some helpful advice to ensure you meet expectations without going on for too long.While many experts will tell you to keep your talk to 3 minutes or less, Chertoff says the best wedding speeches usually last three to five minutes. Gottsman agrees, saying "The perfect speech lasts around 5 minutes, give or take a minute or two."

Just remember: While it's easy to write a lot about the people that you love, there are many festivities that take place during the wedding reception, such as special dances and cutting the cake.

To make sure you're in the sweet spot time-wise, try writing down everything you'd like to say and time yourself as you read. If you end up talking for too long, you'll then want to cut out parts that have less impact.

Practicing is also important because it allows you to see how fast you tend to read. You'll want to make sure you "read slowly, pause, look up, smile, look at the newlyweds and the guests," Blais Comeau says. "Don't rush it."

The debate over whether it's OK to read your wedding speech ends here. Since not everyone is great at public speaking, experts say it's fine to bring notes with you, so long as you're familiar with your script. The important part is to show your love for the couple.

"No one should feel obligated to memorize a speech," Chertoff says. "That being said, it's important that anyone reading a speech or making a toast practice before the wedding day, so they are comfortable and confident on the big day."

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