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10 Things Every Molecular Biologist Should Know PDF
10 Things Every Molecular Biologist Should Know PDF
Every
Molecular
Biologist
Should Know
Nick Oswald
Suzanne Kennedy
Megan Hogan
Megan Cartwright
Edited By Nick Oswald
a BitesizeBio.com eBook
Preface
Preface
Molecular biology is a funny old One of the core aims of Bitesize Bio website yourself, or if you’d just like to let
business. The ton of theoretical (www.bitesizebio.com) is to make it us know what you think (good or bad!) of
knowledge that we cram in during our easier to learn on the job as a molecular our efforts, please feel free to get in touch
undergrad years is scant preparation biologist by providing a place where via the contact page at BitesizeBio.com.
for life at the bench and leaves a lot to these nuggets of vital, often over-looked
be said. knowledge can be found.
Thanks for reading.
When we first hit the lab there are so This short eBook provides 10 such
many things to learn before we even nuggets, neatly contained in one place.
get started that many things go From How SDS-PAGE works to the
unlearned. chemical reason why enzymes have
optimal temperatures we hope that there
How specific techniques work, what The Bitesize Bio team.
will be something in here that will
actually is in those kits we use and why
enlighten and entertain even the most
we use certain approaches rather than
experienced scientist.
others.
If you like the information you find in this
Often these nuggets of knowledge can
eBook - which is the first of many we’ll
mean the difference between
be creating on a variety of topics - we
understanding where an experiment is
would love you pass the file onto your
going wrong and not understanding,
friends to help spread the word about
between getting a result and not getting
what we are doing at Bitesize Bio.
one.
And if you are hungry for more please be
But normally, these nuggets can’t be
sure to visit us at BitesizeBio.com to get
found in lectures or in text books.
the latest articles and eBooks we have
Instead they must be gleaned through
on offer.
hard on-the-job experience or passed
down by word-of-mouth from more Finally if you have any suggestions for
experienced mentors. articles you’d like to see on Bitesize Bio,
if you’d like to write something for the
ii
How SDS-PAGE Works
“If you are still wondering why the stacking gel is needed, think of
what would happen if you didn’t use one.”
migration proportional to
molecular weight.
iv
SDS also coats the protein with a The gel matrix
be made up at a variety of concentrations
uniform negative charge, which masks to produce different pore sizes which, in
the intrinsic charges on the R-groups. turn, can produce varying separating
SDS binds fairly uniformly to the linear In an applied electrical field, the SDS- conditions that can be adjusted to suit your
proteins (around 1.4g SDS/ 1g protein), treated proteins will now move toward needs.
meaning that the charge of the protein the cathode at different rates depending
is now approximately proportional to it’s on their molecular weight. These The discontinuous buffer
different mobilities will be exaggerated
molecular weight. system and the stacking gel
by the high-friction environment of a gel
SDS is also present in the gel to make matrix.
sure that once the proteins are
To conduct the current through from the
linearized and their charges masked, As the name suggests, the gel matrix
anode to the cathode through the gel, a
they stay that way throughout the run. used for SDS-PAGE is polyacrylamide,
buffer is obviously needed. Mostly we use
which is a good choice because it is
The dominant factor in determining the the discontinuous Laemmli buffer system.
chemically inert and, crucially, can easily
migration rate of an SDS-coated protein
“Discontinuous” simply means that the
in a gel is it’s molecular radius.
buffer in the gel and the tank are different.
SDS-coated proteins have been shown
Typically, the system is set up with a
to be linear molecules, 18 Angstroms
stacking gel at pH 6.8, buffered by Tris-
wide and with length proportional to
HCl, a running gel buffered to pH 8.8 by
their molecular weight, so the molecular
Tris-HCl and an electrode buffer at pH 8.3.
radius (and hence their mobility in the
The stacking gel has a low concentration of
gel) is determined by the molecular
acrylamide and the running gel has a
weight of the protein. Since the SDS-
higher concentration capable of retarding
coated proteins have the same charge
the movement of the proteins.
to mass ratio there will be no differential
migration based on charge.
So what’s with all of those different pH’s?
Well, glycine can exist in three different
The discontinuous buffer system
charge states, positive, neutral or negative
v
depending on the pH. This is shown in All of the proteins in the gel sample have So the glycine front accelerates past the
the diagram below. Control of the an electrophoretic mobility that is proteins, leaving them in the dust.
charge state of glycine by the different intermediate between the extreme of the
The result is that the proteins are dumped
buffers is the key to the whole stacking mobility of the glycine and Cl- so when
in a very narrow band at the interface of the
gel thing. the two fronts sweep through the
stacking and running gels and since the
sample-well the proteins are
So here’s how the stacking gel works. running gel has an increased acrylamide
concentrated into the narrow zone
When the power is turned on, the concentration, which slows the movement
between the Cl- and glycine fronts.
negatively-charged glycine ions in the of the proteins according to their size, the
pH 8.3 electrode buffer are forced to And they’re off! separation begins.
enter the stacking gel, where the pH is
6.8. In this environment glycine What was all of that about?
switches predominantly to the This procession carries on until it hits the
zwitterionic (neutrally charged) state. running gel, where the pH switches to
If you are still wondering why the stacking
This loss of charge causes them to 8.8. At this pH the glycine molecules are
gel is needed, think of what would happen
move very slowly in the electric field. mostly negatively charged and can
if you didn’t use one.
migrate much faster than the proteins.
The Cl- ions (from Tris-HCl) on the
Gel wells are around 1cm deep and you
other hand, move much more quickly in
generally need to substantially fill them to
the electric field and they form an ion
get enough protein onto the gel. So in the
front that migrates ahead of the glycine.
The separation of Cl- from the Tris absence of a stacking gel, your sample
would sit on top of the running gel, as a
counter-ion (which is now moving
band of up to 1cm deep.
towards the cathode) creates a narrow
zone with a steep voltage gradient that Rather than being lined up together and
pulls the glycine along behind it, hitting the running gel together, this would
resulting in two narrowly separated mean that the proteins in your sample
The effect of buffer pH on glycine
fronts of migrating ions; the highly would all enter the running gel at different
charge
mobile Cl- front, followed by the slower, times, resulting in very smeared bands.
mostly neutral glycine front.
vi
So the stacking gel ensures that all of
the proteins arrive at the running gel at
the same time so proteins of the same
molecular weight will migrate as tight
bands.
Separation
Once the proteins are in the running Did you find this useful?
gel, they are separated because higher
molecular weight proteins move more
Visit BitesizeBio.com for many more
slowly through the porous acrylamide
eBooks, articles, webinars and videos on
gel than lower molecular weight
topics just like this one.
proteins. The size of the pores in the
gel can be altered depending on the
size of the proteins you want to
separate by changing the acrylamide
concentration.
vii
How Proteases and
Protease
Inhibitors Work
ix
Inhibitors: You can’t stop and can be anything from small enhanced affinity for metal ions to complex
molecules like sodium fluoride to big with them so that nobody else can react
with just one honkin’ polypeptide analogues of with them. This matters because
evolution’s own microbial inhibitors. metalloproteases need zinc, magnesium,
All that chemical lysis, scraping, They can compete with substrates for an manganese, calcium, or even cobalt in their
sonication, and freeze-thawing you active site – sometimes destroying the active site to activate water to hydrolyze
inflict throws the whole system into site in the process – or selfishly hoard peptide bonds to meet their quarterly
chaos. No peptide bond is safe! the precious, precious cations needed hydrolysis goals. Take away that zinc with
for protease function. EDTA, complex up that calcium with EGTA,
Exopeptidases attack the ends of and it’s bye-bye metalloprotease activity.
amino acid chains while Competitive inhibitors act by binding in a
endopeptidases hydrolyze their internal substrate-like manner to the active site to Conclusion: To defeat the
block it or by binding and destroying it
links. Kinases phosphorylate and
through modifications such as protease, you must become
dephosphorylate at will. Good luck
quantifying NOS induction or making a esterification. Many large competitive the protease
case for p53 activation by Western. inhibitors boost their affinity and
Without inhibitors, your results will be specificity by binding to the overlooked
inconsistent, your lab meetings wallflower regions neighboring the hogs- Proteases are just some of the destructive
agonizing, and your graduation date a all-the-glory active site. You have likely forces nature aims at your beloved cell
pipe dream. already come across competitive lysates, and protease inhibitors are one of
inhibitors. They include all sorts, from the ways that the clever scientist can, judo-
Tragically, there is no Wunderkind aprotinin, a 6 kDa polypeptide serine like, turn the universe against itself. For
inhibitor that takes out all proteases, protease that is reversible if you ask more information on beating the universe
halts every phosphorylation event, and politely at pH extremes, to sodium into submission, check out Emily Crow’s
does some light tidying-up about the orthovanadate, a small molecule which “How To Preserve Your Samples In
centrifuges. It takes a “cocktail” of inhibits the ever-gullible protein Western Blotting”, Jode Plank’s “Streamline
approaches to stave off proteolysis. phosphotyrosyl phosphatases. Your Western Blots”, on Bitesize Bio.
Protease inhibitors can act irreversibly,
reversibly, and reversibly-under-duress, By contrast, the toddler-like chelators
(motto: MINE!) take advantage of their
x
Agarose Gels Do Not
Polymerize
Polyacrylamide gel
polymerization
xii
For more information on polyacrylamide So agarose (and agar) gels form by
gel polymerization see Biorad Bulletin gellation through hydrogen bonding
1156 and electrostatic interactions, not
through polymerization.
Agarose gel formation
Chemically, agarose is a
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polysaccharide, whose monomeric unit
eBooks, articles, webinars and videos on
is a disaccharide of D-galactose and
topics just like this one.
3,6-anhydro-L-galactopyranose which
is shown in the diagram below.
Chemically, agarose is a
In aqueous solutions below 35°C these polysaccharide, whose
polymer strands are held together in a monomeric unit is a disaccharide
porous gel structure by non-covalent of D-galactose and 3,6-anhydro-L-
interactions like hydrogen bonds and galactopyranose
electrostatic interactions. Heating the
solution breaks these non-covalent
interactions and separates the strands.
Then as the solution cools, these non-
covalent interactions are re-established
and the gel forms.
xiii
How Ethanol Precipitation of
DNA and RNA
Works
xv
affected by the dielectric constant of the The wash step with 70% vitro translation or reverse transcription. In
solution. these cases, use NaAc.
ethanol…
Water has a high dielectric constant, ▪ Use Ammonium acetate (2M final conc)
which makes it fairly difficult for the Na+ for the removal of dNTPs, but do not use
and PO3- to come together. This step is to wash any residual salt for preparation of DNA for T4
away from the pelleted DNA. polynucleotide kinase reactions as
Ethanol on the other hand has a much
ammonium ions inhibit the enzyme.
lower dielectric constant, making it A few tips on nucleic acid
much easier for Na+ to interact with the To increase the yield in precipitations of
precipitation…
PO3-, shield it’s charge and make the low concentration or small nucleic acid
nucleic acid less hydrophilic, causing it pieces (< 100 nucleotides)
to drop out of solution. Choice of salt
▪ Add MgCl2 to a final concentration of
The role of temperature… ▪ Use Sodium acetate (0.3M final conc, 0.01M
pH 5.2) for routine DNA precipitations
▪ Increase the time of incubation on ice
▪ Use Sodium chloride (0.2M final conc) before centrifugation to 1 hour.
Incubation of the nucleic acid/salt/
ethanol mixture at low temperatures for DNA samples containing SDS since
(e.g. -20 or -80C) is commonly cited in NaCl keeps SDS soluble in 70% ethanol
protocols as necessary in protocols. so it won’t precipitate with the DNA. Did you find this useful?
However, according to Maniatis et al ▪ Use Lithium Chloride (0.8M final conc)
(Molecular Cloning, A Laboratory for RNA. This is because 2.5-3 volumes
Visit BitesizeBio.com for many more
Manual 2nd Edition… 2nd edition?? – I of ethanol should be used for RNA
eBooks, articles, webinars and videos on
need to get a newer version!), this is precipitation and LiCl is more soluble in
topics just like this one.
not required, as nucleic acids at ethanol than NaAc so will not precipitate,
concentrations as low as 20ng/mL will but beware – chloride ions will inhibit
precipitate at 0-4C so incubation for protein synthesis and DNA polymerase
15-30 minutes on ice is sufficient. so LiCl is no good for RNA preps for in
xvi
How Alkaline Lysis Works
including the genomic DNA (gDNA) and This is the selective part. While it is easy
The pellet is then re-suspended in a your plasmid, to single stranded DNA for the the small circular plasmid DNA to re-
solution (normally called solution 1, or (ssDNA). nature it is impossible to properly anneal
similar in the kits) containing Tris, those huge gDNA stretches.
This process is called denaturation and
EDTA, glucose and RNase A. Divalent
is central part of the procedure (which is This is why it’s important to be gentle
cations (Mg2+, Ca2+) are essential for
why it’s called alkaline lysis!). SDS also during the lysis step because vigorous
DNase activity and the integrity of the
xviii
mixing or vortexing will shear the gDNA followed by ethanol precipitation and
producing shorter stretches that can re- affinity chromotography-based methods
anneal and contaminate your plasmid using a support that preferentially binds
prep. to the plasmid DNA under certain
conditions of salt or pH, but releases it
While the double-stranded plasmid can
under other conditions. The most
dissolve easily in solution, the single
common methods are detailed in the
stranded genomic DNA, the SDS and
Bitesize Bio article on 5 ways to clean up
the denatured cellular proteins stick
a DNA sample.
together through hydrophobic
interactions to form a white precipitate.
The precipitate can easily be separated
from the plasmid DNA solution by
centrifugation.
Did you find this useful?
5. Cleaning and
concentration Visit BitesizeBio.com for many more
eBooks, articles, webinars and videos on
topics just like this one.
Now your plasmid DNA has been
separated from the majority of the cell
debris but is in a solution containing
lots of salt, EDTA, RNase and residual
cellular proteins and debris, so it’s not
much use for downstream applications.
The next step is to clean up the solution
and concentrate the plasmid DNA.
“...the downside [of using kits] is that we don't actually know what is in the
mysterious... set of solutions which... makes troubleshooting more difficult.”
xxi
Binding and want to troubleshoot recovery, this The wash steps serve to remove these
can be a step to evaluate further. impurities. There are typically two washes,
although this can vary depending on the
Another way to diagnose problems is to sample type. The first wash will often have
The chaotropic salts are critical for
save the flow-through after binding and a low amount of chaotropic salt to remove
lysis, but also for binding of the nucleic
precipitate it to see if you can find the the protein and colored contaminants. This
acids to the silica resin.
nucleic acids you are searching for. If is always followed with an ethanol wash to
To enhance the binding of nucleic acids you used an SDS-containing detergent in remove the salts. If the prep is something
to silica, alcohol is also added at the lysis, try using NaCl as a precipitant to that didn’t have a lot of protein to begin
binding stage. Most of the time this is avoid contamination of the DNA or RNA with, such as plasmid preps or PCR clean
ethanol but sometimes it may be with detergent. up, then only an ethanol wash is needed.
isopropanol.
Washing Steps Removal of the chaotropic salts is crucial to
The concentration of ethanol, and getting high yields and purity of DNA or
the volume used during binding, both RNA. Some kits will even recommend
have big effects. Too much Your lysate was centrifuged through the washing the column twice with ethanol. If
(concentration or volume) and you’ll silica membrane and now your DNA or salt remains behind, the elution of nucleic
bring in a lot of degraded nucleic acids RNA should be bound to the column and acid is going to be poor, and the A230
and small species that will influence the impurities, protein and reading will be high, resulting in low
UV260 readings and throw off some polysaccharides, should have passed 260/230 ratios.
of your yields. Too little, and it may through.
become difficult to wash away all of the
But, the membrane is still dirty
Dry Spin
salt from the membrane.
with residual proteins and salt. If the
The important point here is that the sample was from plants, there will still be
ethanol influences binding and the polysaccharides, maybe some pigments After the ethanol wash, most protocols
amount added is optimized for too, left on the membrane, or if the have a centrifugation step to dry the
whatever kit you are using. Modifying sample was blood, the membrane might column. This is to remove the ethanol and
that step can help change what you be tinted brown or yellow. is essential for a clean eluant. When 10
recover so if you are having problems mM Tris buffer or water is applied to the
xxii
membrane for elution, the nucleic acids rehydrate in the short time used for Make sure to use fresh high quality ethanol
can become hydrated and will release elution. Elution of DNA can be (100% 200 proof) to dilute buffers or for
from the membrane. If the column still maximized by allowing the buffer to sit in adding to the binding step.
has ethanol on it, then the nucleic acids the membrane for a few minutes before
Low quality ethanol or old stocks may have
cannot be fully rehydrated. centrifugation.
taken on water and not be the correct
Skipping the drying step results in RNA, on the other hand, is fine at a concentration. If the wash buffer is not
ethanol contamination and low yields. slightly acidic pH and so water is the made correctly, you may be washing off
preferred diluent. RNA dissolves readily your DNA or RNA.
The main indication of this problem
in water.
comes when you try to load the sample Low Purity: If the sample is contaminated
onto an agarose gel; the DNA will not with protein (low 260/280) then maybe you
sink, but floats in the buffer, even in the started with too much sample and the
presence of loading dye. Another
What other things can go protein was not completely removed or
indicator of ethanol contamination is dissolved.
that if you put the sample in the -20C, it wrong with silica spin
If the sample has poor 260/230 ratio the
doesn’t freeze. columns? issue is usually salt from the bind or the
Elution wash buffer. Make sure that the highest
quality ethanol was used to prepare wash
Low yields: If you experience yields buffers and if the problem continues, give
The final step is the release of pure lower than you expected for a sample, the column an additional wash.
DNA or RNA from the silica. For DNA there are many factors to think about.
Some samples have a lot more inhibitors
preps, 10 mM Tris at a pH between 8-9 Usually it is a lysis problem, and compared to others. Environmental
is typically used. DNA is more stable at incomplete lysis is a very common cause samples are especially prone to purity
a slightly basic pH and will dissolve of low yields. issues because humic substances are
faster in a buffer. This is true even for
solubilized during extraction. Humics
DNA pellets. Water tends to have a low Alternatively, the problem could be
behave similarly to DNA and are difficult to
pH, as low as 4-5 and high molecular incorrect binding conditions.
remove from the silica column. For this
weight DNA may not completely
type of sample, specialized techniques
xxiii
exist to remove the protein and humics question I ask people is “did you check
prior to the column step. the results of the PCR on a gel?”
because you cannot UV check a PCR
Degradation: This is more of a
reaction and get an accurate reading.
concern for RNA preps and an article
that gives specific advice can be found There is way too much in a PCR reaction
in the Bitesize Bio article absorbing UV at 260: nucleotides,
“Troubleshooting RNA Isolation”. detergents, salts, and primers. In my If you liked this, you’ll
experience, apparent failure of a PCR
Mainly with RNA, degradation occurs
clean-up kit to work frequently is
also like:
from improper storage of the sample or
normally caused by a PCR reaction that
an inefficient lysis, assuming of course
has failed and so there was nothing to
that you eluted with RNase-free water. Recycle Those DNA Extraction
clean up in the first place.
For DNA preps, degradation is not a Columns
huge problem because for PCR, the But if you know you had a strong PCR
DNA can be sheared and it works fine. product, the best approach is to just save by Liam Thompson at BitesizeBio.com
But if you were hoping not to have so your flow-through fraction after binding. If
much sheared DNA, then you may the DNA doesn’t bind, that’s where it is.
have used too strong a lysis method. You can always rescue it and then clean You know those ridiculously priced and
it up again. And then call tech support throw-away DNA mini, midi and maxi-prep
PCR Clean-up Special
and ask for a replacement kit. columns? Well the good news is that you
Considerations: PCR clean-up is the
can actually re-use them if you are
easiest of all silica spin column
reasonably careful at regenerating them,
techniques, because it simply involves
with this simple and cheap method..
adding a high concentration of binding
salts (typically between 3-5 volumes of ...[click to read more]
salt per volumes of PCR reaction) and
centrifuging the column.
xxvi
site must be replenished by ATP.
xxvii
How Colorimetric Assays
Work
One of the great selling points of the Malachite Green Orthophosphate, liberated from a
phosphorlyated substrate upon cleavage
pNPP assay is its simplicity, so it’s easy Assay Mechanism
by the phosphatase, forms a complex with
to see why one might forget that this
The para-nitrophenylphosphate assay is ammonium molybdate in a solution of
bathochromic (a great trivia word!) shift
great, but what if your favorite sulfuric acid. The formation of the
is a crucial element to the assay.
phosphatase doesn’t efficiently malachite green phosphomolybdate
What if, for example, the enzymatic metabolize pNPP? Another commercially complex, measured at 620-650 nm, is
activity requires an optimum pH in the available phosphatase assay is the accordingly directly related to the free
acidic range, as so happens to be the malachite green assay. This simple orthophosphate concentration. Therefore, it
case with acid phosphatase? assay method is based on the complex is possible to quantify phosphorylation and
phosphate release from protein
phosphatase substrates.
xxx
phosphoanhydrides, etc) possess acid- ion associate in the aqueous solution, There’s much more where
labile phosphate groups that can be polyvinylalcohol is added to the solution.
released into solution by incubation at those came from
low pH. On the other hand, lower- Another thing to take into consideration
energy organophosphates during the assay is the possibility of any
These two examples barely skim the
(phosphoesters) are stable in acidic redox reactions that could interfere with
surface of the colorimetric assays. Just
solution and require much harsher the assay. Molybdenum is a transition
think of all the catalase assays for the
conditions (such as thermal metal and so exists in many oxidation
detection of hydrogen peroxide (like
decomposition) to be detected by this states. In the molybdate anion, it has an
Sigma’s quioneimine derived assay), and
method. oxidation state of +6. Reduction of the
of course, the very popular MTT-based
acidified Mo(VI) solution by organic
In the presence of a large excess of assays for measuring cytotoxicity. A better
compounds, like ascorbic acid and
malachite green, the 3:1 ion associate understanding of the chemistry involved in
reducing sugars (i.e. glucose), or by
((MG+)3(PMo12O40 3-)) can easily these assays helps to provide a deeper
inorganic compounds, like SnCl2,
form and precipitate in the acidic understanding of the reactions, as well as
creates the Mo(IV) species, which is blue
aqueous solution. To stabilize the 1:1 any required troubleshooting procedures.
in color.
Colorimetric assays are just one of the
many ways in which chemistry and biology
work hand-in-hand in scientific research. I
think it’s fair to say that the multi-
disciplinary approach is now recognized as
a vital way to move forward in the drug
discovery process.
xxxi
expected to understand pharmacology, Want More Chemistry for Biologists?
cell biology, and molecular biology
techniques. It takes an entire team of
Take a look at this free webinar series from Peter Kirsop at BitesizeBio.com
people, involving many specialities, in
order to be successful in developing
safe and effective drugs. But it is not Chemistry for Biologists
enough simply to involve many
collaborators; I would argue that Covered in this series:
xxxii
How Lab Grade Water is
Purified
xxxiv
(Exactly) Why Enzymes
Have Optimal Temperatures
xxxvii
© 2012 Science Squared Ltd, UK
Photos: Bigstockphoto.com, Sxc.hu
xxxviii