Novagen S

S-protein HRP Conjugate

Description

S-protein HRP Conjugate69047-3

The S•Tag™ System is a protein tagging and detection system based on the interaction

of the 15aa S•Tag peptide with ribonuclease S-protein. This specific binding is of high

affinity (K

d

= 10

-9

M; 1) and allows convenient detection and purification of any protein

fused with the S•Tag sequence (2). Vectors available for making S•Tag fusions are

pET-29a-c(+), pET-30a-c(+), pET-32a-c(+) and pSCREEN™-1b(+) for T7 RNA poly-

merase-driven expression in E. coliand pCITE

®

-4a-c(+) for optimal production of pro-

teins in vitrowith Novagen’s Red Nova

®

Lysate.

The S-protein HRP conjugate is a covalent complex prepared by chemically cross-link-

ing purified S-protein with horseradish peroxidase. The conjugate has been optimized

to give the highest signal:noise ratio when detecting S•Tag fusion proteins even when

present in low abundance in complex protein mixtures. Use of this conjugate allows

single step detection without the need for secondary reagents. The conjugate is highly

specific for S•Tag fusion proteins and can be used in the same incubation mix with

antibodies or steptavidin conjugates. Therefore, when used in conjunction with

Novagen’s Perfect Protein™ Markers, the conjugate becomes a universal marker detec-

tion reagent for any Western blot.

SuperSignal CL-HRP Substrate is available separately for the chemiluminescent detec-

tion of the S-protein HRP conjugate.

Copyright1995 by Novagen, Inc. All rights reserved. S•Tag™, pSCREEN™, pCITE

®

, Red Nova

®

, Perfect Pro-

tein™, the Novagen logo and name are trademarks of Novagen, Inc. SuperSignal is a trademark of Pierce Chem-

ical Company.

Components

•50µlS-protein HRP Conjugate

Storage: Store at -20°C.

Available Separately

Product

Perfect Protein Markers

S•Tag CL-HRP Western Blot Kit

SuperSignal CL-HRP Substrate Kit

25ml 2X Luminol/Enhancer Solution

25ml 2X Stable Peroxide Solution

Size

500µl

50ml

Cat.#

69149-3

69058-3

69059-3

Target Protein Expression

Target genes cloned in pET vectors are commonly expressed by transforming recombi-

nant plasmids into strains that are lysogenic for bacteriophage λDE3. Such strains

carry a chromosomal copy of the T7 RNA polymerase gene under the control of the

lacUV5 promoter. Target protein expression is induced by the addition of IPTG to a

growing culture, which allows production of T7 RNA polymerase and subsequent high-

level transcription of target gene sequences from the T7 promoter.

Detailed protocols for maintenance and induction of pET recombinants are described

in the pET Manual (TB055), which is included with pET Systems and is also available

upon request from Novagen.

Induction of λDE3 Lysogens

After a target plasmid is established in BL21(DE3), HMS174(DE3), NovaBlue(DE3) or

in one of these strains containing pLysS or pLysE, expression of the target DNA is

induced by the addition of IPTG to a growing culture. For pET constructions carrying

the “plain” T7 promoter (N), a final concentration of 0.4mM IPTG is rec-

ommended, while 1mM IPTG is recommended with vectors having the T7lacpromoter

(-29, pET-30 and pET-32). An example of an induction protocol is presented

below.

Pick a single colony from a freshly streaked plate and inoculate 50 ml LB containing

the appropriate antibiotic in a 250ml Erlenmeyer flask. (For good aeration, add medi-

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1

S-protein HRP Conjugate

um up to only 20% of the total flask volume.) Note: include 34µg chloramphenicol/ml if

the cells carry pLysS or pLysE.

Alternatively, inoculate a single colony or a few µl from a glycerol stock into 2ml LB

medium containing the appropriate antibiotic. Incubate with shaking at 37°C until the

OD

600

reaches 0.6-1.0. Store the culture at 4°C overnight. The following morning, collect

the cells by centrifugation (30 sec in a microcentrifuge). Resuspend the cells in 2ml

fresh medium and use this to inoculate 50ml medium.

1.

2.

Incubate with shaking at 37°C until OD

600

reaches 0.4-1 (0.6 recommended;

about 3 hours).

Remove samples for the uninduced control. Add IPTG from a 100mM stock to

a final concentration of 0.4mM (T7 promoter) or 1mM (T7lacpromoter) and

continue the incubation for 2-3hr.

It is often useful to determine where in the cell the target protein is accumulated. Sim-

ple methods for analyzing crude cell fractions are presented below.

Total Cell Protein

The expression of target genes may be quickly assessed by analysis of total cell protein

on an SDS-polyacrylamide gel followed by Coomassie blue staining. Collect induced

cells by centrifugation, resuspend in 1/10 culture volume of 10mM Tris-HCl pH 8.0,

2mM EDTA, remove an aliquot and add to it an equal volume of 2X SDS sample buffer.

Heat to 70°C for 5 min and load 5-20µl on a gel. The proper amount of material to load

depends on the cell density at time of harvest and the expression level of the target

protein. Usually, an amount equivalent to 15µl of a culture with an OD

600

of 1.5 (3µl of

sample by the above method) gives the proper band intensities with Coomassie blue

staining. Much less protein (~1/500 of this amount) is required for Western blot or dot

blot analysis with the S-protein HRP Conjugate.

Soluble and Insoluble Fractions

Many target proteins are expressed in both soluble and insoluble forms. Crude soluble

and insoluble fractions can be prepared by the following protocol. This protocol will

work with any of the pET host strains; in principle the lysozyme addition could be

omitted with strains having pLysS or pLysE.

t induced cells (50ml culture) by centrifugation at 5000 ×g for 5 min.

Discard the supernatant and resuspend the cell pellet in 1/10 culture volume

(5ml) of 50mM Tris-HCl pH 8.0, 2mM EDTA.

Add lysozyme to a concentration of 100µg/ml; use a 10mg/ml stock freshly

prepared in the buffer used in Step 1. Then add 1/10 volume (0.5ml) 1% Triton

X-100. Incubate at 30°C for 15 min.

Place the tube in an ice bath and sonicate with a microtip to shear the DNA.

The solution should lose viscosity after one or two 10 sec pulses at a high out-

put setting.

Centrifuge at 12,000 ×g for 15 min at 4°C. The supernatant contains soluble

proteins; add an equal volume of 2X SDS sample buffer to a sample for gel

analysis. The pellet is the insoluble fraction; resuspend in 1X SDS sample

buffer for gel analysis. An amount of sample corresponding to 30µl of the

original culture volume is usually sufficient for bands to be visualized by

Coomassie blue staining. A 50 to 500-fold dilution provides sufficient protein

for detection by the S-protein HRP Conjugate in most cases.

2.

3.

4.

Western Blot Protocol

an SDS-polyacrylamide gel of the S•Tag fusion sample. Load 5µl of the

Perfect Protein Western Markers (included in the S•Tag CL-HRP Western

Bloy Kit) in one lane. This lane should give bands at 15,000, 25,000, 35,000,

50,000, 75,000, 100,000 and 150,000 daltons when detected with the S-protein

HRP Conjugate. A 5µl load contains 10ng of each species, except the 50,000

dalton band, which is 2X the others (20ng). The 150,000 and 15,000 dalton

bands may give weaker signals than the other species depending on the per-

centage gel, membrane support and transfer conditions used. Due to its large

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S-protein HRP Conjugate

size, the 150,000 dalton band may not transfer completely, and is present at a

significantly lower molar amount than the others. The 15,000 dalton band may

not bind well to the membrane (particularly 0.45µpore size nitrocellulose)

due to its small size.

er the proteins to nitrocellulose electrophoretically. Any standard

device can be used according to the manufacturer’s instructions. The recom-

mended standard transfer buffer is 12mM Tris base, 96mM glycine, pH 8.3,

20% methanol.

Prepare blocking solution by dissolving 1.25g Non-fat dry milk in 25ml

1X TBST (10mM Tris-HCl, pH 8.0, 150mM NaCl, 0.1% Tween 20) with stirring.

This is the amount needed for a typical 7 ×8.5cm blot.

Remove the nitrocellulose from the blotting apparatus and incubate in block-

ing solution at room temperature for 15-30 minutes to block excess protein

binding sites.

Prepare 250ml 1X TBST per blot. Rinse the membrane for 1 minute in

1X TBST at room temperature to remove excess blocking reagent.

Incubate the membrane with a 1:5000 dilution of S-protein HRP Conjugate in

1X TBST for 30 minutes at room temperature.

Wash the membrane 5 times in 25-50ml 1X TBST at room temperature. This

can be done in 1-2 minutes by adding the wash solution, briefly shaking, and

decanting. It is important to thoroughly wash the membrane at this point to

achieve maximum signal : noise ratios.

3.

4.

5.

6.

7.

Detection

Chemiluminescent Detection

e the SuperSignal substrate working solution by combining equal parts

2X Luminol/Enhancer and 2X Stable Peroxide Solution and mixing briefly. For

a typical 7 ×8.5cm blot, 1ml of working solution is sufficient for detection.

Make sure that the entire surface of the membrance has been wetted with the

working solution. Incubate the blot in the working solution at room tempera-

ture for 1 minute.

Remove the membrane from the working solution and place in a plastic sheet

protector or wrap in plastic film. Remove any bubbles between the plastic and

the membrane. Gently remove any liquid from the exterior of the plastic.

Place blot in a film cassette with autoradiographic film and expose for 1-10

minutes. Be careful not to move the film after initial placement or multiple

images can result. An initial exposure time of 1 minute is recommended.

Longer exposures can be performed although the highest light output occurs

in the first five minutes. Light output continues over several hours.

2.

3.

Colorimetric Detection

e a stock solution of 4-chloro-1-naphthol by dissolving 0.3g 4-chloro-1-

naphthol in 10ml ethanol. This solution is stable for at least 1 year if stored

frozen at -20°C. Prepare a 4-chloro-1-naphthol working solution by adding

0.05ml stock solution to 5ml 50mM Tris-HCl, pH 7.6. Remove the white precip-

itate that forms by filtration through Whatman #1 filter paper. Add 5µl 30%

H

2

O

2

.

For a typical 7 ×8.5cm blot, 5ml of working solution is sufficient for detec-

tion. Make sure that the entire surface of the membrance has been wetted

with the working solution. Incubate the blot in the working solution at room

temperature with agitation until color develops, about 30 minutes.

Stop the reaction by rinsing the blot in 1X PBS.

2.

3.

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S-protein HRP Conjugate

Notes:

assure the maximum signal : noise ratio for chemiluminescent or col-

orimetric detection it is important to use non-fat dry milk. Do not substitute

gelatin.

g steps should be performed in sufficient volume and repeated 5

times to assure complete removal of unbound conjugate. If background is

evident on the blot, it can be washed several additional times and more sub-

strate added.

not use azide as a is an inhibitor of horserad-

ish peroxidase and can result in low sensitivity.

Dot blot protocol

a culture of the desired recombinant and prepare a crude protein

extract as described previously. Make serial dilutions of the extract in 10mM

Tris-HCl, 25mM EDTA, pH 8.0 covering a range of 2µg/ml - 200µg/ml protein

(make two additional series for the Perfect Protein Westen Markers and a

negative control (such as E. colilysate made from cells that do not carry the

target plasmid rabbit reticulocyte lysate, etc.;optional).

Spot 1µl samples directly onto nitrocellulose. Allow to air dry for several

minutes.

For detection, proceed as described in the previous section.

2.

3.

References

ds, F.M. and Wyckoff, H.W. (1971) in“The Enzymes”, Vol. IV (Boyer, P.D.,

Ed.), pp. 647-806, Academic Press, New York)

, J.-S. and Raines,R.T. (1993) Protein Science2, 348-356)

, E. and Lane, D. (1988) in“Antibodies: a Laboratory Manual”, Cold Spring

Harbor Laboratory Press, Cold Spring Harbor, New York

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