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FEATURE ARTICLE
IN THIS ISSUE
codon
fraction in
all genes
fraction
in Class II
Arg
Arg
Arg
Arg
Arg
Arg
Gly
Gly
Gly
Gly
Ile
Ile
Ile
Leu
Leu
Leu
Leu
Leu
Leu
Pro
Pro
Pro
Pro
AGG
AGA
CGG
CGA
CGU
CGC
GGG
GGA
GGU
GGC
AUA
AUU
AUC
UUG
UUA
CUG
CUA
CUU
CUC
CCG
CCA
CCU
CCC
0.022
0.039
0.098
0.065
0.378
0.398
0.151
0.109
0.337
0.403
0.073
0.507
0.420
0.129
0.131
0.496
0.037
0.104
0.104
0.525
0.191
0.159
0.124
0.003
0.006
0.008
0.011
0.643
0.330
0.044
0.020
0.508
0.428
0.006
0.335
0.659
0.034
0.055
0.767
0.008
0.056
0.080
0.719
0.153
0.112
0.016
ARTICLES
Overcoming the codon bias of E. coli
for enhanced protein expression
1
Enhanced sensitivity of ChIP
through the application of
Pellet Paint Co-Precipitant
4
Simple, efficient extraction of protein
from hepatocytes with
CytoBuster Reagent
5
TECHNICAL NOTES
Sf9 insect cells and serum-free
media adapted for different
baculovirus applications
7
Western blotting and immunohistochemistry using the HisTag
Monoclonal Antibody
8
NEW PRODUCTS
10
FEATURE ARTICLE
continued from page 1
proL tRNA
leuW tRNA
metT tRNA
pRARE
o ri
Cam
p 1 5a
4694 bp
pLysSRARE
7393 bp
thrT tRNA
glyT tRNA
ileX tRNA
tyrU tRNA
thrU tRNA
lac
I
argW tRNA
Ly
sS
argU tRNA
pLacIRARE
6320 bp
Derivation
Key Feature(s)
Antibiotic
Resistance
Available as
Competent Cells
Tuner
(B)
Cam
Cam
Cam
Cam
yes
yes
yes
yes
RosettaBlue
RosettaBlue(DE3)
RosettaBlue(DE3)pLysS
RosettaBlue(DE3)pLacI
NovaBlue
(K-12)
Tet + Cam
Tet + Cam
Tet + Cam
Tet + Cam
yes
yes
yes
yes
Rosetta-gami
Rosetta-gami(DE3)
Rosetta-gami(DE3)pLysS
Rosetta-gami(DE3)pLacI
Origami
(K-12)
yes
yes
yes
yes
inNovations 12
Compatible vectors*
Rosetta
RosettaBlue
Rosetta-gami
E. coli promoter
based vectors, e.g.
tac, trc, T5,
Rosetta(DE3)
Rosetta(DE3)pLysS
RosettaBlue(DE3)
RosettaBlue(DE3)pLysS
Rosetta-gami(DE3)
Rosetta-gami(DE3)pLysS
Rosetta(DE3)pLacI
RosettaBlue(DE3)pLacI
Rosetta-gami(DE3)pLacI
BL21
(DE3)
I
U
Rosetta
(DE3)
I
U
The authors thank Dr. Jon Beckwith for providing the 6-175 vtPA gene used in this article.
Product
REFERENCES
Rosetta
(DE3)pLacI
I
I
Cat. No.
Price
0.4 ml 70953-3
1 ml 70953-4
Size
$63
$116
0.4 ml 71058-3
1 ml 71058-4
$63
$116
0.4 ml 71054-3
1 ml 71054-4
$63
$116
Rosetta(DE3)
Competent Cells
0.4 ml 70954-3
1 ml 70954-4
$63
$116
Rosetta(DE3)pLysS
Competent Cells
0.4 ml 70956-3
1 ml 70956-4
$63
$116
0.4 ml 71059-3
1 ml 71059-4
$63
$116
0.4 ml 71034-3
1 ml 71034-4
$63
$116
0.4 ml 71055-3
1 ml 71055-4
$63
$116
$63
$116
Rosetta(DE3)pLacI
Competent Cells
0.4 ml 70920-3
1 ml 70920-4
$63
$116
0.4 ml 71060-3
1 ml 71060-4
$63
$116
0.4 ml 71056-3
1 ml 71056-4
$63
$116
Rosetta
Competent Cells
RosettaBlue
Competent Cells
Rosetta-gami
Competent Cells
RosettaBlue(DE3)
Competent Cells
B. pTriEx-3 vtPA
BL21
Rosetta
(DE3)pLysS (DE3)pLysS
M
I
U
I
U
BL21
(DE3)pLacI
I
I
RosettaBlue(DE3)pLysS
Competent Cells
I induced
U uninduced
M markers
vtPA
Rosetta-gami(DE3)
Competent Cells
inNovations 12
RosettaBlue(DE3)pLacI
Competent Cells
Rosetta-gami(DE3)pLacI
Competent Cells
ARTICLE
Victoria Lunyak1, Ryan Baidya2 and Rob Burgess3, 4 1Howard Hughes Medical Institute, La Jolla, CA, 2BioZak/Technology
Developing Center for Gene Therapy Research, San Diego, CA, 3University of California at San Diego, Department of Medicine,
La Jolla, CA, 4TransGenetics, Inc., La Jolla, CA
hromatin Immunoprecipitation
(ChIP) has been successfully
implemented for the analysis of
in vivo proteinDNA complex
formation (1). The technique involves
crosslinking of complexes in living cells
and/or tissues followed by extraction of the
complexed material, subsequent immunoprecipitation with an antibody against the
desired protein and finally PCR to determine the presence or absence of the protein
of interest in a particular region of the
genome. ChIP has been utilized for the
study of numerous transcription factors and
DNA binding proteins with respect to the
target genes regulated by these factors. The
technique is widely applicable, and can be
utilized to conduct studies on both nonDNA binding coregulatory factors such as
NCoR, which has been shown to be involved in regulating hematopoiesis (2), as
well as those which interact directly with
target loci to dictate, for example, specific
transregulatory events during organogenesis, an example of which is Pit-1 (3). It has
also been employed for the characterization
of heterochromatic organization through
studies of methyl DNA binding proteins
and their presence on heterochromatinized
DNA in vivo (4).
Although ChIP may be used in a wide
variety of contexts with respect to the study
of chromatin organization in general and
gene regulation in particular, some applications of the technology require the isolation
and characterization of DNA templates
from increasingly limited amounts of tissue
or considerably heterogenous cell populations of which only a small percentage may
actually contain the desired proteinDNA
complex. Recovery of the necessary
amounts of DNA templates from these
sources needed for efficient PCR is therefore often difficult as much of the starting
material is lost during the extraction and
immunoprecipitation procedure. Novagens
Pellet Paint Co-Precipitant circumvents
As mentioned above, the ChIP methodology entails a step by step procedure for
the isolation and characterization of proteinDNA adducts from living tissue
and/or cell lines. Fig. 1 outlines this procedure. Briefly, the sample in question is
cross-linked via the addition of formaldehyde in order to obtain the intensive network of DNA-protein biopolymers. Crossin vivo cross-linking
adduct recovery
sonication
immunoprecipitation
cross-linkage reversal
Proteinase K treatment
Pellet Paint Co-Precipitant application
for ethanol precipitation
PCR amplification
analysis of PCR products
Figure 1. Chromatin Immunoprecipitation
Assay (ChIP)
inNovations 12
Pellet Paint
use of Pellet Paint did not result in an increase of background or random DNA acquisition, as the quality of the samples obtained with or without the co-precipitant did
REFERENCES
Product
Pellet Paint
Co-Precipitant
Size
Cat. No.
Price
inNovations 12
ARTICLE
continued from page 5
Extract preparation and Western
analysis
kDa
150
100
75
50
CYP3A2
35
25
REFERENCES
CytoBuster Protein
Extraction Reagent
Size
Cat. No.
Price
50 ml
250 ml
71009-3
71009-4
$40
$155
inNovations 12
TECHNICAL NOTE
45
40
35
30
25
20
15
10
5
0
0h
RP
48 h
RP
combination
is far using
superior
for co-transfeca TriEx baculovirus
different
cell/medium
combinations
tion
with linear
baculovirus DNA (data not
A high titer stock of a pTriEx-4/BacVector-3000 baculovirus reshown).
combinant was used to infect cultures of Ready-Plaque Sf9 Cells
The reasons
forMedium
these(RP)
differences
not
in BacVector
Insect Cell
and TriEx Sf9are
Cells
in
TriEx
Insect
Cell
Medium
(T).
The
clone
expresses
E.
coli
clear; however, it is likely that the altered
galactosidese from the AcNPV p10 promoter. Cell samples were
media
formulations cause changes in exharvested immediately after infection (0 h) and at 48 h post-infecpression
that were
result
in the
observed
tion (48 h). patterns
Total cell extracts
assayed
for -gal
activity
using Novagens BetaRed
Kit and results plotted
as arbitrary
phenotypes.
The phenotypes
appear
to be
absorbance units normalized for protein concentration. Equivalent
stable
the were
adapted
cells.
In general,
amounts ofinprotein
also analyzed
by SDS-PAGE
followedwe
by
Coomassie blue staining
recommend
using(inset).
the Ready-Plaque Cells/
BacVector Medium combination for establishment of baculovirus recombinants and
conventional plaque assays, and TriEx
Cells/TriEx Medium for all other applications of the baculovirus expression system.
Application
Product
Size
Ready-Plaque
Sf9 Cells
6 vials
70033-3 $147
BacVector
Insect Cell Medium
1 liter
70590-3
$62
3 vials
71023-3
$68
1 liter
71022-3
$62
inNovations 12
5 assays
70850-3 $145
TECHNICAL NOTE
B. HisTag AP LumiBlot
8
10
10
kDa
225
150
100
75
50
35
25
15
10
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Figure 1. Colorimetric and chemiluminescent Western blot detection of HisTag fusion proteins
BL21(DE3) cells were transformed with appropriate pET vectors encoding proteins with the HisTag sequence in an N-terminal, internal, or C-terminal configuration. Cultures were treated with IPTG for 3 hours to
induce expression and extracts were produced by sonication. Samples from induced cultures were combined with a 10X protein excess of uninduced culture extracts prior to loading. Insect cells and mammalian
COS-1 cell extracts were made with CytoBuster Extraction Reagent. Samples (~ 5 g protein) were loaded on triplicate 420% SDS-polyacrylamide gels and run under constant voltage. Proteins were transferred from the gels to nitrocellulose membranes for Western detection (Panels A and B) or stained with Coomassie blue as indicated. Western detection was performed using a 1:1000 dilution of the HisTag
Monoclonal Antibody and the respective HisTag Western Reagents Kit. Development times were 5 min for Panel A and 40 sec for Panel B.
inNovations 12
Nuclear staining
inNovations 12
NewProducts
NEW PRODUCTS
10
Product
Size
Cat. No.
Price
HT96 NovaBlue
Competent Cells
1 plate
4 plates
20 plates
71011-3
71011-4
71011-5
$375
$1400
$5600
HT96 BL21(DE3)
Competent Cells
1 plate
4 plates
20 plates
71012-3
71012-4
71012-5
$375
$1400
$5600
71031-3
$65
ColiRollers
Plating Beads
Size
Cat. No.
Price
1 pkg
5 pkg
71013-3
71013-4
$7.50
$30
inNovations 12
No transfection
GeneJuice
Toxicity comparison
Three replicate COS-7 cultures were left untreated, transfected
with GeneJuice, and transfected with a popular cationic lipid
based transfection reagent according to recommended protocols.
Cellular damage is visualized by rounding up and detachment
from the plate surface. The photographs, taken 48 h post transfection, show that GeneJuice caused much less cytotoxicity than the
other reagent.
COS-7
HeLa
40
$85
$180
$1440
3.0
2.5
2.0
1.5
GS
GP
1.0
0.5
L2
20
GeneJuice
GS
GP
L2
GeneJuice
Price
GS
60
2.0
1.0
Cat. No.
70967-5
70967-3
70967-4
3.5
3.0
80
GP
4.0
Size
0.3 ml
1 ml
10 1 ml
HeLa
5.0
4.5
4.0
L2
6.0
COS-1
100
RLU/ml (x 10-3)
7.0
RLU/ml (x 10-3)
RLU/ml (x 10-3)
NIH-3T3
GeneJuice
Transfection Reagent
5.0
120
8.0
GeneJuice
9.0
Product
inNovations 12
11
NEW PRODUCTS
gently extracted from E. coli without exposure to heat or oxidative damage and viscosity
is eliminated by nucleic acid digestion in a
single step. The resulting protein extract
can easily be fractionated by conventional
purification techniques. BugBuster HT is
ideally suited for application in high
throughput protein purifications.
BugBuster 10X Protein Extraction
Reagent is a concentrated formulation of
the proprietary detergents employed in
BugBuster without the addition of salts or
buffer components. Concentrated
BugBuster provides a flexible alternative to
the ready-to-use standard 1X BugBuster, allowing user-defined dilution and addition
of buffer components. BugBuster 10X has
all of the bioprocessing benefits of standard
BugBuster plus the freedom to control pH,
reagent concentration, and buffer additives
necessary for maximum extraction and activity of your target protein.
Cat. No.
Price
Size
$80
$295
$750
10 ml 70921-3
50 ml 70921-4
100 ml 70921-5
$42
$184
$350
BugBuster
(primary amine-free)
Extraction Reagent
100 ml 70923-3
500 ml 70923-4
$47
$189
12
Product
HisBind Columns
(resin is pre-charged with Ni2+)
HisBind Magnetic
Agarose Beads
(resin is pre-charged with Ni2+)
Ni-NTA Buffer Kit
Size
Cat. No.
Price
pkg/5
pkg/25
70971-3
70971-4
$85
$340
2 ml
10 ml
71002-3
71002-4
$80
$320
70899-3
$68
inNovations 12
and vortex mixing are not required to generate extracts. Reportasol disrupts the plasma
membrane, leading to mild release of soluble reporters without the need for
freeze/thaw, sonication, or other treatments.
The Reportasol formulation has been
specifically optimized for maximal firefly luciferase, Renilla luciferase, and
Reportasol
Extraction Buffer
Size
Cat. No.
Price
25 ml
5 x 25 ml
70909-3
70909-4
$35
$140
350
3.5
3.0
A570 (BetaRed)
-gal
300
250
2.5
200
2.0
150
1.5
100
50
1.0
0.5
0
Reportasol
Competitor A
Competitor B
FLuc
RLuc
staining with minimal background. The exceptional staining seen with BetaBlue enables quick, accurate determination of
transfection efficiencies, assessment of stable
cell line generation, and transgene expression in tissue slices or whole mounts of
transgenic animals.
Product
Size
Cat. No.
Price
71074-3
$75
COS-1
inNovations 12
13
NEW PRODUCTS
Product
Size
Cat. No.
Price
Mobius 200
Plasmid Kit
25 rxn
70970-3
$165
pkg/25
pkg/25
71019-3
71018-3
$110
$35
70855-3
$50
10 rxn
70969-3
$185
Mobius 1000
Columns
pkg/10
pkg/25
70849-3
70849-4
$95
$225
ClearSpin Filters
pkg/25
70848-3
$45
SpinPrep Kits for rapid plasmid minipreps, gel extraction and PCR clean-up
The SpinPrep PCR Clean-up Kit is designed for the rapid purification of DNA
amplified in PCR reactions. The 10-minute
procedure involves addition of a binding
buffer followed by adsorption of the DNA
to a silica membrane in a spin column
format. Following a wash step, the DNA is
eluted in TE buffer. This kit removes
DNA polymerases, dNTPs, salts, and
> 99% of primers so that they do not interbp
2000
1500
1000
750
500
300
150
50
14
1
2
3
4
PCR Markers
crude PCR product
purified PCR product
PCR Markers
Product
Size
Cat. No.
Price
100 rxn
70976-3
$99
Introductory SpinPrep
PCR Clean-up Kit
20 rxn
70975-3
$25
40 rxn
71073-3
$45
SpinPrep PCR
Clean-up Kit
inNovations 12
Trail Mix Protein Markers for visible tracking and accurate sizing
of proteins in stained gels and Western blots
Trail Mix
Protein Markers
& Trail Mix
Western Markers
Trail Mix
Protein
Markers
Trail Mix
Western
Markers
225
150
100
75
50
35
25
16
15
10
Coomassie blue
420% SDS-PAGE
AP Western Blot
detection (STag)
Product
Trail Mix
Protein Markers
Size
Cat. No.
Price
100 lanes
70980-3
$90
Trail Mix
AP Western Blot Kit
25 blots
71047-3
$110
Trail Mix
HRP Western Blot Kit
25 blots
71048-3
$110
50 l
69598-3
$84
69047-3
$84
Trail Mix
Western Markers
70982-3
$45
S-protein AP Conjugate
25 lanes
7
1
2000
1500
12,000
8000
6000
4000
3000
1000
500
inNovations 12
Size
Cat. No.
Price
100 U
500 U
2,500 U
71003-3
71003-4
71003-5
$45
$195
$950
$140
NovaTaq
DNA Polymerase
71007-3
$140
DNA fragments 0.5 kbp to 7.35 kbp in size were amplified using 2.5 units
NovaTaq DNA Polymerase in a standard 100 l reaction. Products from each
amplification reaction were analyzed by agarose gel electrophoresis (1.2% TAE).
10 mM dNTP Mix
71004-3
$38
0.2 ml
15
ANNOUNCEMENTS
NEW WEB SITE WITH ONLINE ORDERING
On May 7, 2001 Novagen went live with our new web site as part of the CN Biosciences family.
The new site features online ordering, advanced search capabilities and direct access to over 15,000
life science research products, including those from our partner brands Calbiochem, Novabiochem
and Oncogene Research Products. Novagens widely accessed technical resources and vector information are still just a few clicks away. We appreciate your feedback on our new site!
ING
COMO
SO N
PAID
MADISON, WI
PERMIT NO. 370
Novagen, Inc. 601 Science Dr. Madison, WI 53711
16
inNovations 12