Short report

Material and Methods/ Results

DNA methods
Transformation
Minipreps
Enzymatic digestions
Maxipreps
Transformation of expression strains

Protein methods

Whole organism – Drosophila

Protein extraction method
Gel electrophoresis
Imunoblotting

Bacterial strains
Protein expression induction
Protein purification
Affinity chromatography
 DNA methods

Transformation

The plasmids pET30-hsp22 and pET30-hsp22 were transformed into the E.coli
strain DH5, which is one of the hosts for cloning according to the “pET System Manual”
from Novagen. A rapid transformation protocol, which consists in thawing a 100 μl
aliquot of the DH5α competent cells on ice, adding 1 μl of the plasmid, incubating the
tubes on ice for 20 minutes, heat pulsing then in a 42ºC water bath for 45 seconds and
reincubating them on ice for 2 minutes was used. After that 900 μl of Soc medium were
added and incubated at 37ºC for 30 minutes with shaking at 225 – 250 rpm. At this point
LBagar – Kanamicin plates had been prepared. 100 μl were plated into one plate using a
sterile spreader and the remaining 900 μl were plated in another plate. These were
incubated at 37ºC overnight.

Minipreps

Using the plates resulting from the previous step, one single colony was isolated,
inoculated into 2 ml of LB – Kanamicin and allowed to grow at 37ºC overnight. It was
done for both plasmids, with 4 replicates (from 4 different colonies) each.
An alkaline lysis protocol was used to extract the plasmids from the cells and a
1% agarose gel electrophoresis was performed (the gel was stained with Etidium
Bromide). (Figure 1).
 A B C D M E F G H

Figure 1: Agarose Gel Electrophoresis. Lanes A, B, C and D have the

four replicates of pET30-22 transformed into DH5α. Lanes E, F, G and F have
the four replicates of pET30-27 transformed into DH5α, and M is the lane
with a 1 Kb marker.

Some plasmid extractions were also done with the QIAprep Spin Miniprep Kit
Protocol using a micro centrifuge, from Qiagen, because the DNA resulting from the
Alkaline Lysis method seemed to resist to digestion with restriction enzymes.

Enzymatic Digestions

The DNA was first digested with the enzyme XhoI in a total volume of 50 μl (at
this step 20 μg/ml of RNAse were added). The enzymatic reaction was incubated at 37ºC
for 2 hours. Then 20 μl were used to do a 1% agarose gel electrophoresis (Figure 2). The
remaining DNA was precipitated with ethanol and ressuspended it in 20 μl of water
before the second digestion, this time with the enzyme HindIII. This step was necessary
because these enzymes have different buffers, which means the second enzyme couldn’t
be mixed with the first reaction. The second digestion was also made in a total volume of
50 μl, and incubated at 37ºC for 2 hours. After this a 1% agarose gel electrophoresis was
made (Figure 3).
 A B C D M E F G H
Figure 2: Agarose Gel Electrophoresis of the DNA digested with
XhoI. Lanes A, B, C and D have the four replicates of pET30-22. Lanes E, F,
G and F have the four replicates of pET30-27, and M is the lane with a 1 Kb
DNA Ladder (Gibco).

A B C D M E F G H
Figure 3: Agarose Gel Electrophoresis of the DNA digested with XhoI
and HindIII. Lanes A, B, C and D have the four replicates of pET30-22. Lanes
E, F, G and F have the four replicates of pET30-27, and M is the lane with a 1
Kb DNA Ladder (Gibco).
 It seems like there was no second digestion, which makes sense if one looks at the
pET30 system map (Novagen) in Appendix I. If indeed the cDNAs were inserted
between the NcoI and the XhoI sites, the HindIII site was gone, and none of the inserts
has one. Based on that map several digestions were planned:

1. 2 hours digestion with XhoI (37ºC) followed by a 2 hours digestion with

BglII (37ºC). This digestion should cut of the insert in both plasmids. The approximate
expected sizes would be:
pET30-hsp27 ≈ 5400
≈ 600
pET30-hsp22 ≈ 5400
≈ 500
2. 2 hours digestion with XhoI (37ºC) followed by a 2 hours digestion with
NcoI (37ºC). This digestion should cut of the insert only in pET30-hsp27. The
approximate expected sizes would be:
pET30-hsp27 ≈ 5400
≈ 600
pET30-hsp22 ≈ 5900

3. 2 hours digestion with SmaI (25ºC). This digestion should cut pET30-
hsp22 once and pET30-hsp27 twice. The approximate expected sizes would be:
pET30-hsp27 ≈ 4400
≈ 1600
pET30-hsp22 ≈ 5900

4. 2 hours digestion with EcoNI (37ºC). This digestion should cut pET30-
hsp22 twice and pET30-hsp27 three times. The approximate expected sizes would be:

pET30-hsp27 ≈ 3700
≈ 2000
≈ 300
 pET30-hsp22 ≈ 3700
≈ 2200

Figure 4 shows the result of these digestions in a 1% agarose gel electrophoresis.

A B C D E M F G H I J M
Figure 4: Agarose Gel Electrophoresis of the DNA digested with
different restriction enzymes. Lane A – pET30-22 digested with XhoI and
BglII; Lane B – pET30-22 digested with XhoI and NcoI; Lane C – pET30-22
digested with SmaI; Lane D – pET30-22 digested with EcoNI; Lane E – non
digested pET30-22; Lane F – pET30-27 digested with XhoI and BglII; Lane G
– pET30-27 digested with XhoI and NcoI; Lane H – pET30-27 digested with
SmaI; Lane I – pET30-27 digested with EcoNI; Lane J – non digested pET30-
27 and M is the lane with a 1 Kb DNA Ladder (Gibco).

For some reason the DNA didn’t behave like expected. The decision was made to
proceed with the Maxi prep anyway.

Maxi prep

One colony of the transformed cells was isolated, inoculated into 2 ml of LB –

Kanamicin and allowed to grow for about 16 hours at 37ºC, both for the DH5α-pET30-
hsp22 and for the DH5α-pET30-hsp27. Afterwards, these pre-inoculums were inoculated
into 1L of LB-Kanamicin and incubated overnight at 37ºC. The DNA was purified
according to Promega’s “Technical Bulletin”. The concentration of the purified DNA was
calculated using its absorbance at 260 nm, and its purity was measured by the
(Absorbance at 260)/(Absorbance at 280) nm ratio. Hsp27 was at a concentration of 30.6
mg/ml and hsp22’s concentration was 8.43mg/ml. They were considered pure, since the
(Absorbance at 260)/(Absorbance at 280) nm was approximately 1.80 in both.

Transformation into expression strains

The strains BL21DE3 and GJ1158 were made competent and transformed in the
same way as described for DH5α, except that GJ1158 had to be plated in LBONagar-
Kanamicin instead of LBagar-Kanamicin. DNA Minipreps were performed before
inducing the protein expression, and a 1% agarose gel electrophoresis was made(Figure
5)

Figure 5: Agarose Gel Electrophoresis. Lane A –

pET30-hsp22 from the maxiprep; Lane B – pET30-
hsp27 from the strain GJ1158; Lane C – pET30-hsp22
from the strain BL21DE3; Lane D – pET30-hsp27 from
the maxiprep; Lane E – pET30-hsp22 from the strain
GJ1158; Lane F – pET30-hsp27 from the strain
BL21DE3. M is a molecular weight ladder (λ Hind III –
Gibco)

M A B C D E F

Whole organism – Drosophila

 Protein extraction method
Approximately 70 flies were stressed by placing them inside 50 ml Falcon tubes
in a water bath at 29ºC for 20 minutes and then at 36ºC for 100 minutes. Half of that
sample was given a recovery time of about 16 hours. They were all frozen in liquid
nitrogen until the moment of extraction. The control sample was frozen without being
exposed to the stress conditions.
The samples were added 500 µl of 1% SDS , boiled during 5 minutes,
homogenized in a Potter device, boiled for another 5 minutes and briefly centrifuged.
The amount of protein in the samples was calculated by using the BCA method
(BCA protein assay kit - Pierce®).
A 15% SDS-PAGE was then done, and loading 20 µg/µl of protein in each lane.
The run was done in a Biorad Mini-protean device. Part of this gel was dyed with
Coomassie Blue and the rest was transferred into a nitrocellulose transfer membrane.

Figure 6: SDS-PAGE (15%) of protein extracts from

D. melanogaster stained with Coomassie Blue. Lane A: heat
shocked sample; lane B: heat shocked sample with 16 h
recovery; lane C: non heat shocked sample; Lane D is a
positive control (S2 heat shocked, sent by you) and M is a lane
with a molecular weight marker (Kaleidoscope Prestained
standards - Biorad)

ABCMD

An imunoblotting assay was performed with these membranes using the α− hsp22
and the α− hsp27 antibodies that you’ve sent (primary antibody); anti mouse IgG –
alkaline Phosphatase conjugate (Sigma) and monoclonal anti rabbit imunoglobulins –
alkaline Phosphatase conjugate (Sigma) were used as secondary antibodies, the last step
being done with BCIP/NBT color development substrate (Promega).

a b

M A B C D M D C B A

Figure 7: Imunoblot from D. melanogaster homogenates. a) Probing

with α-hsp27 antibody; b) Probing with α-hsp22 antibody; Lane A: heat
shocked sample; lane B: heat shocked sample with 16 h recovery time; lane C:
non heat shocked sample; Lane D is a positive control (S2 heat shocked, sent
by you) and M is a lane with a molecular weight marker (Kaleidoscope
Prestained standards - Biorad).

Bacterial strains

Protein expression induction

One isolated colony of GJ1158-pet30-hsp22 and another one of GJ1158-pet30-

hsp27 were inoculated in 2 ml of LBON-Kan each; BL21-pet30-hsp22 and BL21-pet30-
hsp27 were also inoculated in 2 ml of LB-kan. These were all grown at 37ºC overnight.
Afterwords, these pre-inoculum were poured into 100 ml of fresh KB-Kan or LBON-
Kan, depending on the strain. They were incubated at 37ºC until the OD at 600 nm had
reached 0.6. The protein induction was performed as recommended in the protocol
you’ve sent us. The cells were then centrifuged at 3000g for 10 minutes, and the pellet
was ressuspended in 5 ml of a 1% SDS solution. The samples were boiled during 5
minutes, sonicated, boiled again for another 5 minutes and briefly centrifuged. One
microliter of each of these samples was loaded into a 15 % SDS-PAGE, to check if the
induction was working. Part of this gel was dyed with Coomassie Blue (Figure 8) and the
rest of it was transferred into a nitrocellulose transfer membrane. This membrane was
then used in an imunoblotting assay, with the antibodies α-hsp27 and α-hsp22 (Figure 9)
 Figure 8: SDS-PAGE (15 %) of cell extracts resulting from the
induction of protein expression. Lane M: molecular weight marker
(Kaleidoscope Prestained standards - Biorad); Lane A: GJ1158 transformed
with pet30-hsp22; Lane B: BL21 DE3 transformed with pet30-hsp22; Lane C:
GJ1158 transformed with pet30-hsp27.

MABC
a b

A B C D E F M A B C D E F

Figure 9: Imunoblot of the samples resulting from protein expression

induction a) Probing with α-hsp27 antibody; b) Probing with α-hsp22
antibody; Lane A: GJ1158-pet30-hsp27 without induction; Lane B: BL21
DE3-pet30-hsp22 without induction; Lane C: GJ1158-pet30-hsp27 induced
with NaCl; Lane D: GJ1158-pet30-hsp22 induced with NaCl; Lane E: BL21
DE3-pet30-hsp27 induced with IPTG; Lane F: BL21 DE3-pet30-hsp22
induced with IPTG and M is a lane with a molecular weight marker
(Kaleidoscope Prestained standards - Biorad).

After realising the induction had been successful, the induction was repeated at a
larger scale, with 500 ml of culture medium.

Protein purification

All the purification steps were performed at 4º C, in a refrigerator, according to

the protocol you’ve sent. Fractions were collected from all chromatography steps, and a
SDS-PAGE (12%)was performed and the resulting gels were dyed with Coomassie Blue.
The gels were scanned in “GS710 calibrated imaging densitometer – Biorad” and the
images treated with the “Quantity one 4.2.1” software. The following images are
examples of the gels obtained form the different samples.

a)

A B C D E F G M

b)
 A B C D E F G M

c)

A B C D E F G M

d)
 A B C D E F G M

Figure 10: SDS PAGE (12%) of all the fractions resulting from the
purification procedure for different samples. a) BL21 DE3-pet-hsp22; b) BL21
DE3-pet-hsp27; c) GJ1158-pet-hsp27; d) GJ1158-pet-hsp22. Lane A: Cell As
lysate; Lane B: Flow throught; Lane C: Wash with Binding Buffer; Lane D:
Wash with Washing Buffer ; Lane E: Elution; Lane F: Column wash with
Strip Buffer; Lane G: Dialysate and M is a molecular weight marker
(prestained low range molecular weight marker - Biorad)
you can see, the bands corresponding to the dialysate fraction didn’t seem very sharp,
which is why a 2D PAGE on two sampleswas performed, GJ1158-pet30-hsp22 and
GJ1158-pet30-hsp27. These showed that the purificatins hadn’t been completely
successful (Figure 11). Therefore a re-purification was made, by dialysing the eluted
fraction in Binding Buffer and repeating the whole purification procedure. The eluted
fraction from this purification was again loaded in a 2D PAGE and was shown to be pure
(Figure 12)

a)
b)

Figure 11: 2D PAGE of the eluted fractions, dyed with silver stain. a)
GJ1158-pet30-hsp22; b) GJ1158-pet30-hsp27. At the right you can see the
molecular weight marker (Low molecular weight marker - Amersham)
 Figure 12: 2D PAGE of the re-purified eluted fraction of GJ1158-
pet30-hsp22, dyed with silver stain. At the right you can see the molecular
weight marker (Low molecular weight marker - Amersham)

Affinity chromatography

We are now adapting two affinity chromatography protocols in order to find out
which proteins interact with hsp22 and hsp27. One of the protocols uses the same buffers
as the purification. The protein is bound to the resin, washed, Drosophila’s homogenate
passed through the column, washed and eluted. The other approach uses AC buffer (10%
glicerol, 100mM NaCl, 20mM Tris pH 7.6, 0.5mM EDTA, 0.1 % Tween) more or less in
the same way. The chromatographies done so far don’t show a big amount of protein in
the eluted fraction (Figure 13)

A B C D E F G H I M
Figure 13: SDS PAGE(12%) of all the fractions resulting from the
affinity chromatography, stained with Coomassie Blue. Lane A: Cell lysate;
Lane B: Flow throught; Lane C: Wash with Binding Buffer; Lane D: Wash
with Washing Buffer ; Lane E: Drosophila homogenate; Lane F: Flow
through; Lane G: wash with Washing Buffer; Lane H: elution; Lane I: wash
with Strip Buffer and M is a molecular weight marker (prestained low range
molecular weight marker - Biorad)