Biochemistry Lab Techniques for the MCAT: Everything You Need to Know
/Learn key MCAT biochemistry techniques, plus practice questions and answers
Part 1: Introduction to biochemistry lab techniques
Part 2: Gel electrophoresis
a) Basic principles
b) Types
a. Native-PAGE
b. SDS-PAGE
c. Reducing SDS-PAGE
d. Isoelectric focusing
Part 3: Blotting methods
a) Basic principles
b) Types
a. Western blot
b. Southern blot
c. Northern blot
Part 4: DNA-based techniques
a) DNA sequencing via Sanger method
b) Polymerase chain reaction
a. RT-qPCR
b. cDNA library
Part 5: Enzyme-linked immunosorbent assay (ELISA)
a) Basic principles
b) Types
a. Direct
b. Indirect
c. Sandwich
Part 6: Molecular-biology techniques
a) Basic principles
b) Molecular cloning
c) Bacterial transformation
Part 7: Centrifugation and Chromatography
a) Basic principles
a. Gel filtration (size exclusion) chromatography
b. Ion-exchange chromatography
c. Affinity chromatography
Part 8: Biochemistry lab techniques high-yield terms
Part 9: Biochemistry lab techniques practice passage and answers
Part 10: Biochemistry lab techniques practice standalone questions and answers
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Part 1: Introduction to biochemistry lab techniques
Welcome to our guide on experimental techniques in biochemistry. This is a high-yield topic, and a knowledge of the experimental techniques we will discuss will help you when you take your MCAT. One of the most difficult parts about learning these techniques is that they’re often presented at a very complex level, but we’ll provide concise and clear explanations in this guide. In many cases, it’s also easy to feel like you need to learn everything about an experimental technique to master these questions on the MCAT, but we’ll show you exactly what you need to know!
This is a long guide, but it’ll help you with the complex biochemistry techniques that the MCAT will throw at you. Remember, the MCAT test-writers develop passages by adapting scientific articles and asking you questions. By understanding these techniques, you’ll put yourself in the position to answer the diverse array of questions you may be asked on the exam.
We’re going to go into many of the techniques that may show up on your MCAT, including chromatography, molecular cloning, DNA sequencing, PCR, Blotting, ELISA, and gel electrophoresis. We’ll focus on the details that will help you ace these questions from an MCAT perspective, and we’ll finish with some sample questions to help you assess your proficiency.
(Suggested reading: MCAT Biochemistry: Everything You Need to Know)
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Part 2: Gel electrophoresis
a) Basic principles
Gel electrophoresis is an experiment used to separate different components of a mixture based on their size and charge. Normally, these components are strands of DNA, RNA, or different proteins. Let’s say you have a test tube containing 5 different proteins, but you only want one of them. If the 5 proteins each have a different size (or different amino acids), you can separate them with gel electrophoresis.
During an experiment, you begin by placing your mixture on the gel, which you can think of as a large sheet of Jell-O. You then apply an electric field across the gel using a negatively charged side (cathode) and a positively charged side (anode). The molecules will travel through the Jell-O, and you can think of the system as a molecule swim meet through a Jell-O pool. (Note: many gel electrophoresis experiments are referred to with the acronym PAGE, or polyacrylamide gel electrophoresis. Polyacrylamide just refers to the type of gel that is used!)
All gel electrophoresis experiments work by taking advantage of three properties: size, charge, and shape.
1. Size
Let’s look at size first: pretend each of our 5 proteins has the same net negative charge. When you turn on the electric field, all 5 proteins will move towards the positively charged side of the electric field. However, the proteins are in the Jell-O, so if one protein is really big, it’ll move more slowly than the smaller proteins. (Think of walking through waist-deep water versus knee-deep water!) If you start the race by turning on the electric field, all the proteins will move towards the finish line, but the smallest will get there first. If you stop the race at any point by turning off the electric field, your proteins will stop moving.
2. Charge
While molecules are usually separated by just size, you need to remember that charge can also be a factor. Let’s say we start with 3 proteins of equal size. Protein 1 has a net charge of -5. Protein 2 has a net charge of -20. Protein 3 has a net charge of -40. Which protein will travel towards the positively charged anode more quickly? Protein 3 will travel towards the positive end of the gel faster than Proteins 1 and 2 because it is more highly charged and experiences greater attraction to the positive side. On the other hand, protein 1 will travel the slowest.
As we saw above, the total charge does vary for proteins, and this variation of charge is dependent on the protein’s side chains. Acidic side chains are negatively charged when deprotonated, while basic side chains are positively charged when protonated. If you add up all of these charges, you get a net charge that’ll tell you 1) how quickly the protein will move and 2) in which direction.
How does charge affect gel migration for nucleic acids? You might know that in the structure of DNA and RNA, each nucleoside or subunit (A, C, G, T, or U) is joined to the next using a link that contains a phosphate group. Importantly, each phosphate group carries a negative charge, so DNA and RNA will always be negative and will always be attracted to the positive charge. As a result, you can assume that every strand of DNA and RNA has a constant and equal distribution of charge or an equal charge density. For this reason, charge isn’t a factor when comparing how quickly different strands of DNA and RNA move, and it is only dependent on their size and shape.
3. Shape
The last big factor is molecule shape or aerodynamics. The more aerodynamic or streamlined a substance is, the faster it will move through the gel. Think of a race car that weighs the same amount as a school bus; the race car is more aerodynamically designed than the bus, so the race car still should travel faster than the school bus even if they weigh the same amount.
b) Types
a. SDS-PAGE
Since the charge density throughout a protein can vary, separating proteins is a little more complex than separating DNA or RNA. While smaller DNA and RNA strands will almost always travel faster than larger strands, proteins may break this general rule of thumb if they have different charge densities.
For example, you might have a large protein with a lot of negative side chains and a slightly smaller protein with fewer negative side chains. Will the larger protein travel faster because it is more negatively charged? Or will the smaller protein travel faster because it is smaller? Even for similarly sized and charged proteins, the 3D structure of the protein may vary a lot, meaning aerodynamics are another factor we might have to deal with.
In order to eliminate the effects of the differences in charge distribution and 3D shape for proteins that we mentioned above, researchers use SDS-PAGE. In other words, if you want to separate proteins just by their size (number of amino acids), use SDS-PAGE!
In SDS-PAGE, researchers add sodium dodecyl sulfate (SDS) to their proteins before running them on the gel. SDS denatures the protein and adds a number of negative charges that are proportional to the size of the protein, thereby creating an equal charge distribution (just like we see in DNA and RNA).
You can think about protein denaturing as untying a difficult knot. The primary structure of a protein is the string that was used to create the knot, and the secondary, tertiary, and quaternary structures were created when you made that terrible knot, which is often necessary for protein function in our analogy. Adding SDS unties the knot, so you are left with an untied string containing a number of negative charges that are proportional to the length of the string. The shorter strings will always travel faster than the longer strings, and we no longer have to worry about charge!
Figure: SDS-PAGE gel
b. Reducing SDS-PAGE
There’s one small thing that we haven’t told you about SDS-PAGE yet: adding SDS completely denatures (or straightens) the protein except at places where there are disulfide bonds. Disulfide bonds are formed by the connection between two different cysteine side chains of a protein, and you can think of them as taping two points on a string together. This might cause the protein to travel more slowly than if these two points weren’t attached.
In order to break these disulfide bonds, you have to add a reducing agent that reduces the single disulfide S-S bond to two S-H bonds. This is known as reducing SDS-PAGE. By using reducing SDS-PAGE, you ensure that all of the higher structure of a protein has been eliminated, including any disulfide bonds.
c. Native-PAGE
Sometimes you want to analyze a protein in its natural state, but once a protein is denatured, it is often impossible to return it back to its normal or native shape (imagine untying a massive knot and then being told to re-tie that exact knot!). If you want to isolate a protein and conduct a study that would require the protein to be in its native shape (e.g., studying your protein’s activity after adding an inhibitor), you need to use native-PAGE. In native-PAGE, you do not add SDS or reducing agents, and the gel is non-denaturing, so the protein can remain in its native shape and maintain its secondary, tertiary, and quaternary structure.
Figure: Native, SDS-PAGE, and Reducing SDS-PAGE gels
d. Isoelectric focusing
In the 3 electrophoresis techniques we’ve discussed, you introduce your molecules near the negatively charged side (cathode) and watch them migrate towards the positively charged side (anode). However, we’ve already discussed that the net charge on proteins may also be neutral or even positive. If the net charge was positive, the protein would run the wrong way!
Let’s illustrate this problem by saying we have 3 proteins. Protein 1 has three negatively charged side chains (e.g., two aspartates and one glutamate) and one positively charged side chain (e.g., lysine). Let’s say protein 2 is completely neutral. And let’s say protein 3 has three positively charged side chains (e.g., lysine, arginine, and histidine). Protein 1 has a net charge of -2. Protein 2 has a net charge of 0. And Protein 3 has a net charge of +3.
If you place all 3 proteins on the negative end of the gel, only the negatively charged protein (Protein 1) will move (towards the positive side), and you wouldn’t have separated proteins 2 and 3. To solve this problem, we use a technique called isoelectric focusing.
Isoelectric focusing is similar to our previous experiments, except that there is a stationary pH gradient (ranging from about 0 to 14) inside the gel in addition to the electric field. The lowest pH is found near the positive side of the gel (anode), and the highest pH is found near the negative side of the gel (cathode). If you add components to this gel, they will migrate until reaching a region where the pH is equal to their isoelectric point, which is known as the pI. The isoelectric point is the pH at which the protein has a completely neutral charge.
How do we determine the isoelectric point of a protein? Let’s go back to those acidic and basic side chains. Acidic side chains will be deprotonated and negatively charged at a high pH. As you decrease in pH, however, more and more of these side chains will be protonated. In fact, most acidic side chains will be protonated by the time you get to a pH of about 2.
Basic side chains, on the other hand, will be protonated and positively charged at low pH. As you increase pH, more and more of these side chains will become deprotonated. Most basic side chains will be deprotonated by a pH of about 12. So, as you increase the pH, negative charges are gained whereas decreasing pH results in gaining positive charges.
The isoelectric point is the pH at which the number of negative charges is equal to the number of positive charges and your protein has a net neutral charge. Once your protein is neutrally charged, it will stop moving, and it won’t be attracted to the positive or negative poles of the gel!
Figure: Isoelectric focusing gel
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