Sie sind auf Seite 1von 24

Bio 332

Lab #2 Yeast molecular biology: epitope tagging of a protein.


(Adopted from Knop et al, 1998 & Current Protocols in Molecular Biology, 2003)
Saccharomyces cerevisiae (baker’s yeast) is often considered to be a great model
eukaryotic organism in a manner analogous to Escherichia coli as a model prokaryotic
organism. Yeasts are just as easy to grow as other microorganisms, and they have a haploid
nuclear DNA content only 3.5 times that of E. coli. Despite the small genome sizes, these
yeasts display most of the features of higher eukaryotes. The fact that many cellular
processes are conserved among different eukaryotic species––combined with the powerful
genetic and molecular tools that are available––has made these yeasts important experimental
organisms for a variety of basic problems in eukaryotic molecular biology.
Culturing yeast is simple, economical, and rapid, characterized by a doubling time of
approximately 90 mins on rich medium (such as YEPD). Budding yeast cells divide
mitotically by forming a bud, which pinches off to form a daughter cell. The progression
through the cell cycle can be monitored by the size of the bud; this has been used to isolate a
large collection of mutants (called cdc mutants) that are blocked at various stages of the cell
cycle. Since yeast can be grown on a completely defined medium, many nutritional
auxotrophs have been isolated. This has not only permitted the analysis of complex
metabolic pathways but has also provided a large number of mutations useful for genetic
analysis.
Yeast can exist stably in either haploid or diploid states. A haploid cell can be either
of two mating types, called a and α. Diploid a/α cells – formed by fusion of an α cell and
an a cell – can grow mitotically indefinitely, but under conditions of carbon and nitrogen
starvation will undergo meiosis. The meiosis products, called spores, are contained in a
structure called an ascus. The haploid yeast cell has a genome size of about 15 megabases
and contains 16 linear chromosomes, ranging in size from 200 to 2200 kb. Thus, the largest
yeast chromosome is still 100 times smaller than the average mammalian chromosome.
Aside from different media requirements, yeast cells are physically manipulated
essentially as described for bacterial cells – i.e. they are grown in liquid culture (in tubes or
flasks) or on the surface of agar plates and are manipulated using the basic equipments
you’ve learned in your previous laboratory courses. Wild-type S. cerevisiae grows well at

14
30°C with good aeration and with glucose as a carbon source. This is in contrast with E. coli
which grows at 37°C. When using culture tubes, vortex the contents briefly after inoculation
to disperse the cells. Erlenmeyer flasks work well for growing larger liquid cultures. For
good aeration, the medium should constitute no more than one-fifth of the total flask volume,
and growth should be carried out in a shaking incubator at 250 rpm.
The genome of the yeast S. cerevisiae was the first eukaryotic genome that has been
completely sequenced (Goffeau et al, 1996). This fact, together with its easy genetic
manipulability, makes bakers’ yeast a highly versatile unicellular eukaryotic system to study
basic cellular processes. PCR-based methods exist which allow gene targeting for either
deletion-replacement or insertion of heterologous DNA at discrete loci on the genome,
allowing a fast functional analysis of genes (Baudin et al, 1993). Such an approach to
targeted disruption or tagging of genes is particularly useful for systematic analysis of several
genes in parallel. As many yeast genes have homologues in higher eukaryotes, many
research groups not primarily focused on work with yeast cells became interested in studying
the function of these genes in yeast. The success of this work largely depends on the
availability of easily applicable methods to manipulate yeast strains. In this laboratory
exercise, we will use a PCR-generated module for the C-terminal tagging of yeast
chromosomal genes (see diagrams A & B on the next page) and employ the characteristics of
these affinity tags to demonstrate protein-protein interactions via co-immunoprecipitation
experiment; which is a simple and effective method for detecting or confirming a physical
interaction between two proteins in vivo. The amount of extract and antibody necessary for
efficient and effective immunoprecipitation can vary greatly and has to be determined
empirically for each experiment.

Tagging of the yeast Hmt1 protein


The S. cerevisiae Hmt1 protein is a protein arginine methyltransferase that catalyzes
the methylation reaction of arginine residue(s) within the context of a protein. This post-
translational modification is found on many cellular proteins and plays an important role in
RNA processing and gene expression steps. Biochemical data indicate that Hmt1’s
biochemical association can be affected by the loss of Ada2, which is a component of the S.
cerevisiae SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex.

15
SAGA complex is a multifunctional activator that regulates transcription by RNA
polymerase II. This complex is responsible for allow the transition from a compact and
inactive chromatin state to open and active chromatin state to take place, which is pre-
requisite for transcription. The role of this complex is to achieve post-translational
modification of the amino terminal tails of histones, which allows the creation of a proper
epigenetic environment for gene expression. The yeast SAGA complex is composed of 21
widely conserved proteins that are organized into functional sub-modules.
To determine how Ada2 affects Hmt1’s biochemical interaction, we will employ a
co-immunoprecipitation approach. In this laboratory, we will assess the role of Ada2 on the
functional biochemical association between Hmt1 and Psp2, a cytoplasmic protein that plays
a potential role in mitochondrial mRNA splicing. To achieve this, we will need to create
some specific yeast strains: PCR will be first used to generate a 9xmyc (9 copies of myc tag)
tagging modules for Hmt1. This DNA module will be transformed into yeast via yeast
transformation; this yeast strain already contains a differently tagged Psp2 (TAP-tagged).
Once the DNA module is inside the yeast cell, homologous recombination takes place
between the flanking sequences of the module and the chromosomal sequences, thereby
allowing this module to be inserted at the C-terminal domain of the HMT1 DNA sequence.
The consequence of which is to allow the expression of a Hmt1 fusion protein that contains
nine copies of myc epitopes at its C-terminal domain (called Hmt1-myc). Two different
yeast strains will be used, both of which already harbor TAP-tagged Psp2 (TAP= Tandem-
Affinity Purification): one of these two strains is a wild-type (ADA2) strain and the other is
an ada2-null strain. We will use an anti-myc antibody to first probe for myc-tagged Hmt1 to
determine whether Hmt1 associates with TAP-tagged Psp2 in a co-immunoprecipitation
experiment. If Hmt1 associates with Psp2, we will then determine whether the absence of
Ada2 is critical for this association.

c-myc tag (from Terpe, Appl. Microbiol Biotechnol, 2003)


c-myc tag can be placed at the N- or C- terminus and it is a widely used epitope tag. The
antibody epitope of eleven amino acids can be expressed in a different protein context and
still confers recognition by the specific immunoglobulin (such as the murine anti-c-myc

16
antibody called 9E10). Myc-tagged proteins can be affinity-purified using antibody-
conjugated agarose beads.
Designing PCR primers for gene-specific tagging of HMT1 gene, using the Knop method
(Knop et al, 1999):
Forward Primer: 5’- gene specific sequence---CGTACGCTGCAGGTCGAC-3’
Reverse Primer: 5’- gene specific sequence—ATCGATGAATTCGAGCTCG-3’ (in reverse
complement – you should know what that means!)

17
Lab 2, Part 1
• PCR – done by the TA in advance, before the class begins.
• Cast a 1% Agarose gel – for each student
PCR reaction set-up
5µl of Primer 1 (10µM concentration; for Hmt1, use MCY28)
5µl of Primer 2 (10µM concentration; for Hmt1, use MCY29)
10µl of NEB Thermopol buffer
2µl of 10mM ea. dNTP mix
1µl of plasmid DNA (100ng/µl), for tagging 9 copies of myc onto HMT1 gene (pYM21 or
MYP324, this will allows 9-myc to confer NatR resistance)
75µl of water
start the PCR reaction - once the PCR reaction reaches 55°C, add the following:
1µl of AmpliTaq (Applied Biosystems)
1µl of PFU turbo (Stratagene)
PCR program is as follows:
Cycle 1: 2 min, 55°C (add AmpliTaq & PFU turbo at this step! – hot start!)
Cycle 2: 5 min, 72°C
Cycle 3: 2 min, 95°C
Cycle 4: 30 sec, 95°C
Cycle 5: 30 sec, 55°C
Cycle 6: 2 min, 72°C
Cycle 7: go to cycle #4, repeat 29 times.
Cycle 8: 10 mins, 72°C
Cycle 9: hold at 8°C.
(total run time for the PCR = 2 hrs & 32 mins)

1. For each student, set up a new microfuge tube on a rack.


2. Once the PCR reaction is finished, the TA will allow you to take 5µl out of the PCR
reaction tube and add these into the microfuge tube on your rack.

18
3. Then add 2µl of 6X DNA loading dye to the microfuge tube containing 5µl of the
PCR product, mix well by gently pipette up & down – BE CAREFUL as to not
introducing any air bubbles!!
4. Load the entire mix (there should be a total of 7µl in your microfuge tube) into a
single well on your 1% agarose gel that you just casted earlier.
5. In a different well, load 10µl of the DNA molecular weight ladder (provided by the
TA).
6. Turn on the power source to 100 volts and electrophorese the gel for 20 mins.
7. Stain the agarose gel on a tray with enough ethdium bromide solution (provided by
the TA) to cover the gel. Gently rock the tray for 20 mins on a rocker.
8. Discard the staining solution into the proper hazardous waste (liquid) container.
Destain the gel with distilled water for 10 mins.
9. With the aid of a TA/instructional support, use the gel doc system to visualize your
DNA bands within the agarose gel. The TA will print your data out – label the wells
and keep this document for your lab report later on.

19
Bio 332
Lab#2, Part 2

On the night before lab, done by the TA:


• Grow both strains of yeast (ADA2 and Δada2) in fresh 5ml YEPD to 1-2 x 107
cells/ml at 30°C for overnight (Done by the TA; can grow in a flask based on the
number of students in the class)
High-efficiency Lithium Acetate Transformation of S. cerevisiae
Alkali cations such as lithium acetate make yeast competent in taking up DNA. The protocol
calls for brief suspension of yeast cells in buffered lithium acetate, followed by the addition
of transforming DNA with carrier DNA (such as salmon sperm DNA). The addition of
polyethylene glycol (PEG) and a heat shock will trigger DNA uptake into the yeast cells.
Adopted from Gietz et al, 1992
1. (TA does this in the morning) Using a new flask, dilute the overnight yeast culture to
2 x 106 cells/ml with fresh YEPD (or a 1:100 dilution), and grow this to 0.5 x 107
cells/ml (approx. 3-4 hrs; done by the TA). 10 ml is needed for each transformation
– the TA should calculate the volume needed based on the number of students in your
class, and increase the total by 30 ml for any potential extra need.
2. Make fresh 1x TE/LiOAc from the 5X stock solution (5X stock solution is provided
by the TA). Label your name on a 15ml centrifuge tube.
3. The TA will aliquot 10ml of the diluted culture (ready for transformation) into your
labeled 15ml centrifuge tube. Spin down the 10ml of cells in this tube using the
clinical centrifuge at high speed for 2 minutes. Discard the supernatant by gently
pouring the liquid down the sink, and wash the cell pellet with 10ml (1x volume) of
sterile water. Close the cap and vortex the tube briefly to mix/wash the cells.
4. Spin down the cells and discard the supernatant as the last step. Using a P1000,
resuspend the cell pellet in 1 ml of sterile water and transfer the entire mix into a
sterile 1.5ml microfuge tube.
5. Spin down the cells in microfuge for 5 secs at 12K RPM. Discard supernatant by
gently pouring out the liquid.

20
6. Wash the cell pellet in 1ml of freshly prepared 1XTE/LiOAc in a manner similar to
step #4.
7. Vortex briefly to resuspend the cell pellet (approx 2x109 cells/ml) in 1X TE/LiOAc.
8. Spin down the cells in microfuge as above and discard supernatant. Resuspend the
cells in 50-75µl (volume depending on the volume of transformed DNA) of 1X
TE/LiOAc. Then add the following:
1 µg plasmid DNA (or PCR products, provided by your TA)
5µl 10mg/ml freshly boiled/chilled ssDNA (maximum transformation
efficiency is achieved by repeating the denaturation cycle (boiling & chilling)
of carrier DNA immediately prior to use).
9. Incubate the microfuge tube on a rolling rocker at 30°C for 30 mins (during this
incubation time, make the mix that contains PEG with LiOAc, needed below in
step#10). Make sure you place your tube in a symmetrical manner with the others.
10. Remove your microfuge tube from the rocker, add 300µl of 40% PEG in LiOAc
(made fresh, 8 parts 50% PEG and 2 parts 5X LiOAc) into your microfuge tube.
11. Vortex the microfuge tube thoroughly (look for homogeneity in viscosity).
12. Incubate the microfuge tube on a rolling rocker at 30°C for another 30 mins.
13. After the 30 minute incubation period, add 35µl of DMSO to the tube, mix by brief
vortexing, and heat shock by placing the tube in the heat block at 42°C for 15 mins.
(longer or shorter duration of the heat shock compromises the transformation
efficiency)
14. **IF YOU ARE USING AN ANTIBIOTIC SELECTION MARKER SUCH AS NAT or
KAN/G418: add the entire mix into a test tube containing 5ml of YEPD, allow the
cells to recover for 3 hrs at 30°C in a rollerdrum, otherwise, go to step #15
15. Spin down the cells in a microfuge for 5 secs at 12K RPM.
16. Remove supernatant, resuspend cells in 100-200µl of sterile TE8
17. Plate the entire volume onto a petri dish containing selective media. Spread the
volume across the entire media surface using glass beads (demonstrated by the TA).
Transformants will be visible 2-5 days after plating, depending on the strain and the
media (rich YEPD vs. synthetic complete, for example) used.

21
Solution Recipes used in Yeast Transformation

5X LiOAc/TE:
0.5M LiOAc, 0.05M Tris(pH 8.0), 0.01M EDTA (pH 7.5),

50% PEG w/v (MW 3350)


Make this in water and sterile filter the solution using a 0.22µM size filter and a sterile
syringe. Prepared by the TA.

22
Bio 332
Lab #2, part 3
• Colony PCR to confirm the genotype of the transformants
• Cast the SDS-PAGE gel to be used later
Polymerase chain reaction (PCR) technology has enabled rapid analysis of DNA
sequences and has opened doors to further scientific inquiry. Because DNA amplification can
occur in the presence of other cellular components such as proteins, RNA and nonspecific
DNAs, it is possible to amplify directly from bacterial cell lysates without first purifying the
DNA. An amplification-compatible reagent is all one needs to lyse the cell and release the
template DNA. Lyse-N-Go PCR Reagent (Pierce, cat#78882) is one such reagent that will
release DNA from cells in a form that is ready for PCR amplification.
Lyse-N-Go Procedure:
1. Aliquot 5µl of the Lyse-N-Go reagent into a thin-wall 0.65ml PCR tube. Repeat this
step for 5 tubes (USE FILTER TIPS FOR PCR PROCEDURES!).
2. Use a sterile P-20 pipette filtered tip, carefully pick small amounts of a colony with
the tip and dip the tip into the 5µl of the Lyse-N-Go (demonstrated by the TA).
Completely resuspend the cells in the 5µl of the Lyse-N-Go reagent by gently
pipetting up and down. Repeat this process for all 5 tubes with 5 different, isolated
colonies from your transformation plate – if you don’t have enough transformants,
use some of your classmates’.
3. Carefully close each tube’s cap, bring the tubes to your TA at the front of the class in
a rack, and the TA will place them in a thermal cycler and run the following program
(it takes approx. 19 mins for the program to run):
i. Cycle 1: 65°C, 30 sec
ii. Cycle 2: 8°C, 30 sec
iii. Cycle 3: 65°C, 90 sec
iv. Cycle 4: 97°C, 180 sec
v. Cycle 5: 8°C, 60 sec
vi. Cycle 6: 65°C, 180 sec
vii. Cycle 7: 97°C, 60 sec
viii. Cycle 8, 65°C, 60 sec

23
ix. Cycle 9, 80°C, hold
4. When the thermal cycle program reaches cycle 9 and on hold, the TA will open up
the cover of the thermalcycler and add 45µl of the PCR mix (set up by the
instructional support or the TA) into each of your five tubes (keep ALL tubes on the
thermocycler at all time). The TA will gently pipette up and down to mix the
reagents for each tube.
PCR Mix (per reaction, for a single colony, 45µl total per reaction)
1µl of primer 1 (10µM concentration)
1µl of primer 2 (10µM concentration)
5µl of 10X Taq Buffer
1µl of Taq
1µl of 10mM dNTP mix
36µl of water

5. Terminate the Lyse-N-Go thermal cycle program on the thermal cycler, and
immediately begin the new thermal cycle program aimed at amplifying the DNA.
6. While the thermal cycle is running the DNA amplification program (which takes
approximately 1.5 hrs), each student will cast a 1.5% agarose gel to run your
samples when the PCR is done.
7. After you had casted your agarose gel, start casting TWO 10% SDS-PAGE gels that
you will be using during later class periods.
8. Once the PCR program is done, take the tubes out, add 6µl of 10X DNA-loading dye
into each tube and mix well by gently pipetting up and down. In each of your agarose
gel lanes, load 10µl of this mix and perform agarose gel electrophoresis on them with
the purpose of examining whether the amplification had worked or not. THINK
ABOUT WHAT A POSITIVE RESULT SHOULD LOOK LIKE! Restreak positive
clones onto a fresh plate. Let them grow at 30°C until Monday.

24
Procedures on casting the SDS-PAGE gels (see gel recipes on pg. 27):
• Follow the manufacturer’s direction to set-up and use the Bio-Rad SDS-PAGE gel
casting system (“Mini-Protean 3 Cell Assembly Guide” – see the next page)
• Use the following recipe for SDS-PAGE gels (10% gel) – pour the resolving gel until
¾ of the way from the bottom, then add a thin layer of isopropanol to shield the gel
away from contacting with air. This allows for even polymerization of the gel. After
the gel has polymerized, pour away the isopropanol above a sink (careful to make
sure you do not release the clip!) and rinse the cavity briefly with water from a
squirt bottle, dab dry with kimwipe as much as you can, then pour the stacking gel
and insert the gel comb (10-well comb).
• When making the gel solution from the recipe below, make sure to add TEMED and
APS the last!
• Once the stacking gel is polymerized, carefully wrap the entire gel with a water-
soaked paper-towel. Do not remove the comb! This is followed by saran wrap for
the entire gel. Write your name on it with a sharpie and store this at 4°C.

25
26
27
Recipes
Resolving gel (for 2 gels):
3.3 ml ProtoGel (National Diagnostics, cat#EC-890, via Biochem Stockroom)
3.3 ml milipore water
2.4 ml 1.5M Tris pH 8.8
100µl of 10% SDS
10µl of TEMED
50µl of 10% APS (Ammonium persulfate)

Stacking gel (5% final), for 2 gels:


650µl of ProtoGel
3.2 ml of water
475µl of 1.5M Tris pH 6.8
37.5µl of 10% SDS
5µl of TEMED
25µl of 10% APS

10x SDS-PAGE Running Buffer


30.3 g Tris base
144.2g glycine
10g SDS
make up to 1L with water

20x Western Transfer Buffer (use at 1X) - for 1L


30.3g Tris base
144.2g glycine
make up to 1L with water

28
Bio 332
Lab #2, part 4
• Making yeast lysates and perform co-immunoprecipitation
The night before the class, performed by the TA
*Innoculate two isolated colonies from each strain onto 2 tubes each containing 50 ml of
YEPD, grow O/N at 30°C, shaking. You’ll have a total of 4 tubes, 2 of which are WT with
Hmt1-myc & Psp2-TAP, and 2 of which are ada2-null with Hmt1-myc and Psp2-TAP.

**Do all the following steps ON ICE! Label EACH microfuge tube with your name on
the side at all steps!
1. Harvest the cells by spinning at 2500 RPM for 3 mins in the clinical tabletop
centrifuge in a 50 ml centrifuge tube. Remove supernatant as shown before.
2. Complete your lysis buffer (1X PBS-MT) with protease inhibitors (PLAAC,
benzamidine, and PMSF) during the centrifugation steps! – Benzamidine (2.6µl per
ml of buffer made), PMSF (5µl per ml of buffer made), and PLAAC (1µl per ml of
buffer made).
PMSF must be made fresh prior to the start of class by the TA – 0.035g per ml of ethanol.
Benzamidine must be thawed on ice and PLAAC must be thawed at room temperature
since DMSO does not thaw on ice.
3. Wash the cells with 50 ml of ice-cold 1X PBS. Centrifuge and discard supernatant as
described in step #1.
4. Repeat the above step. Place the cell pellet containing tube on ice.
5. Use a P1000 to resuspend the cell pellet using the residual PBS inside the 50 ml
centrifuge tube. Transfer the entire suspension to an eppendorf tube. Flash spin in a
microfuge and carefully remove the excess liquid using a pipetteman.
6. Add approximately 100µl of acid-washed microbeads (same volume as pellet) into
the microfuge tube containing the cell pellet. Then add approximately 150µl of the
protease-inhibitor-completed lysis buffer (1X PBS-MT & protease inhibitors).
7. Use the BulletBlunder located in the cold room as per the manufacturer’s setting.

29
8. Place the microfuge tube back on ice, immediate add 500µl of the cold, completed
lysis buffer to the tube. Close the tube and vortex in the highest setting for a couple
of seconds, place the tube back on ice.
9. Centrifuge the microfuge tube for 10 mins at maximum RPM using the microfuge
located inside the cold room.
10. During the 10 min. centrifugation step, wash 30µl of IgG sepharose bead slurry (pre-
aliquoted out by the TA) with 1ml of cold 1X PBS-MT (no protease inhibitor
added). Spin the beads at 2000xg for 1 min in a microfuge at room temperature each
time and remove the supernatant. Repeat this 3 times total.
11. Remove 500µl of supernatant from the microfuge tube after the 10 min spin in the
cold room and place them into another microfuge tube that contains your washed IgG
beads. Place the microfuge with both the lysate and the beads onto a rotator in the
cold room and allow the immunoprecipitation to take place for 1.5 hrs.
12. From the leftover lysate, a small aliquot (~10µl) into another clean microfuge tube
and mix this with 5µl of the 3X SDS-Sample Buffer (label this as your “INPUT”).
Give this to the TA to store at -20°C until the next class.
13. After 1.5 hrs, spin the microfuge tube at 2000 RPM for 30 seconds in the cold room,
remove 10µl of the supernatant into a new tube (label this as your “post-IgG IP”)
and carefully discard the rest of the supernatant. Place this “post-IgG IP” tube on ice.
14. Add 1 ml of ice-cold 1X PBS-MT (completed with protease-inhibitors) to the pelleted
beads, invert the tube 3-4 times to mix and wash the beads.
15. Spin the beads at 2000 RPM for 30 seconds at 4°C, use the P1000 to carefully remove
and discard the supernatant.
16. Repeat the wash for two more times.
17. Use a P200 to carefully remove as much liquid as you can WITHOUT removing any
of the beads – do this under the light to better visualize what you are pipetting out!
18. To the beads, add 25 µl of 3x SDS-sampling buffer and boil for 5 mins. Make sure
you use a cap lock!! Label this tube as “ELUATE” and with your name written on
the side of the tube. Give this tube to the TA to store at -20°C until the next class
period.

30
19. To the “post-IgG IP” tube on ice – mix in 5µl of the 3X SDS-Sample Buffer as before
and give this to the TA (make sure your name is labeled on the side!) to store at -
20°C until the next class.
Solution Recipes:
10X PBS
For 1 liter
80g NaCl
2g KCl
14.4g Na2HPO4
2.4g KH2PO4
fill up to 1 liter with milipore water
1X PBSMT (make 50ml, store in 50ml conicals):
6 mM KCl (use 3M stock solution)
5 mM MgCl2 (use 1M stock solution)
1% Triton X-100 (use 10% stock solution)
1X PBS (use 10X stock solution)
PLAAC (Protease inhibitor cocktail mix): use 1µl per ml of buffer
Contains 2.5µg/ml of each of the following:
Leupeptin
Chymostatin
Antipain
Pepstatin A
Aprotinin
Store at -20°C, but thawed at room temp instead of on ice shortly before use. DMSO
does not thaw on ice.
200 mM PMSF (phenylmethylsulfonyl fluoride), toxic! Use 5µl per ml of buffer
0.035g PMSF per 1ml of 100% ethanol
**Very short half-life; about 1 week, store at -20°C.
0.5M Benzamidine: use 2.6µl per ml of buffer
Dissolve in water. Store at -20°C.

31
Bio 332
Lab #2, part 5
• SDS-PAGE
• Immunoblotting transfer
• Start of the immunoblot
--Take all of your saved samples out of the -20°C storage and place them in your microfuge
tube rack. Add the cap lock to each microfuge tube and place them on the heat block to boil
for 5 mins to denature your protein sample.
--Take your microfuge tubes out of the heat block, remove the cap lock for each one, and
vortex each sample briefly.
--Use the small microfuge centrifuge on your bench to bring down all the liquid to the bottom
of your microfuge tube.

Protocol for loading SDS-PAGE


1) Unwrap the SDS-PAGE gel saved from last session. Rinse the gel gently with
distilled water.
2) Carefully remove the comb by slowly pulling away the comb from the gel – do not do
this too fast as you will rip the wells apart!
3) Assemble the gel onto the apparatus as described in the Bio-Rad protocol shown
previously (and demonstrated by the TA/instructor)
4) Load the gel in the order specified below, using SPECIAL GEL LOADING TIPS!
**You will be given a positive and a negative control lysate to load.

Loading the SDS-PAGE gel, starting from your left to right:


Lanes: 1 2 3 4 5 6 7 8 9 10
Marker Input Post-IP Elute Buffer Input Post-IP Elute (-) CTRL (+) CTRL
Vol.Load: 10µl 15µl 15µl 12µl 15µl 15µl 15µl 15µl 15µl 15µl

---- ADA2 strain ------ ---Δada2 strain -----

5) Once the gel is loaded (each person = one gel), attach the electrodes, turn the
powerpack to 110 volts and monitor the electrophoresis of the stacking gel by
watching the travelling of the dye front.

32
6) Once the dye-front reaches the stacking gel, turn the powerpack up to 185 volts and
let the gel run until the dye-front had ran out of the gel (takes approximately 1-2
hrs).
7) During the electrophoresis of your protein gel, gather the materials you will need to
set-up the transfer of proteins onto nitrocellulose membrane for the immunoblotting
experiment. The TA will demonstrate how you will set up your immunoblot
transfer.
8) Once the gel electrophoresis is finished, use the plastic gel cutter and cut out the
stacking gel. You will use the resolving gel part for immunoblotting (see protocol
below).
Protocol for Immunoblot Transfer (WEAR GLOVES!):
1. Cut nitrocellulose membrane and four pieces of thick 3mm Whatman filter paper, all in
the same size of gel from which samples are to be transferred. Make one liter of 1X transfer
solution.
2. Soak membrane in transfer solution.
3. Set up gel transfer as below – make sure the “black” side of the plastic clamp is at the
bottom. The order (from bottom to the top) is as follows: sponge, Whatman papers (x2), gel,
filter, Whatman papers (x2), sponge. Do this in a glass tray with some 1X transfer buffers in
it (but not too much!).

+ two 3mm filters

nitrocellulose
gel

two 3mm filters


_

4. Roll a ½ size plastic pipet over the tower as each layer is placed in order to

33
avoid bubbles between layers.
5. Placed the clamp together, and insert into the Bio-Rad wet-transfer system. Insert the
icepack from -20°C, then fill the tank to top of the transfer module with more 1X transfer
buffer. Add a small stir bar, put the entire apparatus on a stir plate, connect the electrodes,
and turn the powerpack to run at 100 volts for 45 mins at RT (wait for your classmates to
maximize the spaces within the tank).
6. Once the transfer is completed, shut off the power pack, disconnect the electrodes, and
remove the apparatus away from the stir plate.
7. Using a forcep, remove the filter and place this onto a plastic western tray – be aware of
the side of the nitrocellulose membrane that contacts the gel; place the membrane with this
side UPWARD.
8. Stain filter with Ponceau S to make sure transfer occurred – shake briefly for a few seconds.
9. Pour the stain back to the tube containing Ponceau – this stain can be recycled/re-used!
10. Rinse the blot gently with water to wash away excess Ponceau, you should see bands stained red.
11. Gently wash the stain off with water and gently use a pencil to MARK the molecular
weight ladder on the membrane – look for different coloring patterns!
12. Put copious amounts of the blocking agent (5% milk in 1XPBS-T & sodium azide
(1µl of 10% solution per ml used)).
13. Cover/seal the tray with parafilm!
14. Incubate until the next class with gentle rocking at 4°C.

Ponceau S solution staining of the immunoblot membrane


Ponceau S Solution is a ready–to–use, reversible staining solution designed for rapid
(5 minute) staining of protein bands on nitrocellulose or PVDF membranes (Western
blots), as well as cellulose acetate membranes. Ponceau S stain is easily reversed with
water washes, facilitating subsequent immunological detection.

34
Bio 332
Lab #2, part 6
• Finish immunoblotting (anti-myc, to probe for Hmt1’s presence).
Protocol for Western blotting – continued
**The TA will add primary antibody (α-myc) 1 hr prior to the start of the class.
1. Carefully pour the primary antibody into a 15ml conical tube. They can be saved for
use in the future.
2. Rinse with copious amounts of 1X PBS-T
3. Wash with copious amounts of 1X PBS-T for 5 mins
4. Wash with copious amounts of 1X PBS-T for 15 mins
5. Add secondary antibody to 1X PBS-T/5% milk, (10 ml, 1:5000 dilution)
6. Put on rocker for 1 hr.
7. Rinse with copious amounts of 1X PBS-T
8. Wash with copious amounts of 1X PBS-T for 5 mins
9. Wash with copious amounts of 1X PBS-T for 15 mins
10. Rinse with copious amounts of milipore water
11. Wash with copious amounts of water
12. While the blot is being washed in water, make up your developing solution (ECL;
Pierce cat# p134080): 1 ml total – 500µl of solution 1 and 500µl of solution 2 (1:1
ratio) Make it in an eppendorf tube.
13. Use a P1000, develop the blot by pipetting the developing solution onto the blot for 1
min straight.
14. Use tweezers to handle blot – gently lift the blot and make sure the blot stays moist
but the majority of the liquid does not reside on the blot
15. Place onto a plexiglass, wrap the whole thing with a seran wrap – careful to make
sure the surface is flat rather than wrinkled look.
16. Go to the 6th floor dark room where film developer is, expose the blot to the film: do a
5 sec exposure, then a 1 min exposure.
17. Develop the film in the film developer.
18. Align the film with the alignment signals on the plexiglass, draw the molecular
weight markers on the film.

35
Graphic Diagram of the Principles Behind Immunoblotting:

Once you have marked the film based on its alignment with the membrane, the membrane
will be re-blocked as before using the blocking buffer, and the same membrane will be
probed for the amount of protein A present to determine the relative amount of Psp2 you had
immunoprecipitated.

36
Bio 332
Lab #2, part 7
• Finish immunoblotting (anti-protein A, to probe for amount of Psp2 pulled
down in your immunoprecipitation)

See protocols on immunoblotting from Lab #2, part 6 – complete the immunoblotting
procedure using anti-Protein A antibody!

On Friday
Recitation and discussion of the results.

37

Das könnte Ihnen auch gefallen