TB325 GST Tag Monoclonal Antibody

8/14/2019 TB325 GST Tag Monoclonal Antibody

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GSTTagTM

Monoclonal Antibody

About the Kit

GSTTag Monoclonal Antibody 50 g 71097-3

250 g 71097-4

Description

The GSTTag Monoclonal Antibody is a mouse monoclonal antibody (IgG1) with affinity for the

26 kDa glutathione-S-transferase (GST) domain fromS. japonicum. This highly purified antibody

is superior for detecting GSTTag fusion proteins expressed inE. coli, yeast, mammalian, and in

vitro transcription/translation systems by Western blotting, immunoprecipitation or

immunofluorescence. GSTTag fusion proteins can be efficiently expressed inE. coli using

Novagens pET-41a-c(+) or pET-42a-c(+) vectors. The 50 g kit provides enough antibody for 50

Western blots (10 cm 10 cm).

Specificity glutathione-S-transferase (GST) protein, precise epitope not determined

Species/isotype Mouse monoclonal IgG1

Cross-reactivity Negligible with bacterial, insect or mammalian cell lysates

Sensitivity 2.55 ng: Western blot developed with chromogenic substrates


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GSTTagTM

Monoclonal AntibodyWestern Blotting

Chemiluminescent detection

This protocol is for the transfer and detection of proteins on nitrocellulose membranes. Alkali-

soluble Casein is the recommended blocking reagent because it results in the lowest background.

BSA or gelatin can be used for greater sensitivity but may result in a higher background.

Preparation

1. Prepare fresh blocking solution for washing the membrane. For each standard 10 10 cm

blot, prepare 20 ml of blocking solution. Either dilute 5% Alkali-Soluble Casein prepared with

5X TBS (Cat. No. 70955-3) to 1% with deionized water or prepare a stock of Alkali-Soluble

Casein (Calbiochem Cat. No. 218680) according to Appendix A, page 7. Alternatively prepare

1% Gelatin or 3% BSA in 1X TBST as the blocking solution.

2. Prepare 40 ml fresh blocking solution for the primary and secondary antibody dilutions per

standard 10 10 cm blot. Novagen recommends 0.5% Alkali-Soluble Casein in 0.5X TBS.

Dilute 5% Alkali-Soluble Casein prepared with 5X TBS (Cat. No. 70955-3) to 0.5% using

deionized water. Alternatively, prepare 0.5% Gelatin or 3% BSA in 1X TBST.

3. Prepare 80 ml 1X TBS (150 mM NaCl, 10 mM Tris-HCl, pH 7.5) and 180 ml 1X TBSTT (500

mM NaCl, 20 mM Tris-HCl, 0.05% v/v Tween-20, 0.2% v/v Triton

X-100, pH 7.5) per standard

10 10 cm blot.

4. Dilute the GSTTag Monoclonal Antibody 1:10,000. Dilute 2 l of antibody into 20 ml of

blocking solution.

5. Dilute the Goat Anti-Mouse IgG AP Conjugate (Cat. No. 69266-3) or HRP Conjugate (Cat. No.

71045-3) 1:5,000 in blocking solution. Dilute 4 l of antibody into 20 ml blocking solution.

Protocol

1. The following steps should be performed at room temperature with gentle rocking or

agitation during incubations. Use a clean tray and place the membrane protein-side up.

2. Run a SDS-polyacrylamide gel of the GSTTag fusion protein sample. Load protein markers

in an adjacent lane. Perfect Protein (Cat. No. 69959-3) or Trail Mix (Cat. No. 70982-3)

Western Markers are available from Novagen and require a S-protein Conjugate (AP; Cat. No.

69598-3, or HRP; Cat. No. 69047-3) or the HisTag

Monoclonal Antibody (Cat. No. 70796-3)

for detection.

Note: Detection of Trail Mix Western Markers with the HisTag Monoclonal Antibody and Goat Anti-

Mouse IgG HRP Conjugate (H+L) is not recommended. As an alternative, use Perfect Protein

Western Markers with this conjugate.

3. Transfer the proteins to a membrane electrophoretically. Any standard device can be used

according to the manufacturer's instructions. The standard transfer buffer is 192 mM glycine,

25 mM Tris Base, 20% methanol, pH 8.3. If using the Perfect Protein or Trail Mix Western

markers, the 150 and 225 kDa bands may transfer incompletely due to their large size. The 15

kDa band may not efficiently bind to the membrane (particularly 0.45 pore size

nitrocellulose) due to its small size.

4. Wash the membrane twice for 5 min each time with 20 ml 1X TBS.

5. Incubate for 30 min in 20 ml blocking solution.

Note: Note that PVDF or other hydrophobic membranes may require different blocking conditions (e.g.

longer blocking times, higher concentrations of blocking reagent).

6. Wash twice for 5 min each time using 20 ml 1X TBSTT.

7. Wash for 5 min with 20 ml 1X TBS.

8. Incubate for 1 h with 20 ml GSTTag Monoclonal Antibody diluted 1:10,000 in blocking

solution.

9. Wash twice with 20 ml for 5 min each time using 1X TBSTT.


11. Incubate for 1 h with 20 ml Goat Anti-Mouse IgG AP or HRP Conjugate in blocking solution.

2 TB325 Rev. C 0503

United States & Canada 800-207-0144Germany 0800 6931 000United Kingdom 0800 622935

Or your local sales officewww.novagen.com

Novagen


3/7

GSTTagTM

Monoclonal AntibodyNote: HRP conjugates cannot be used with blocking buffer containing sodium azide. Sodium azide

inhibits HRP activity.

12. Wash five times for 5 min each with 20 ml 1X TBSTT. It is important to thoroughly wash the

membrane at this point to achieve maximum signal:noise ratios.

Note: Washing steps should be performed in sufficient volume and repeated 5 times to assure

complete removal of unbound conjugate. If background is evident, the blot can be washed

several more times before adding additional substrate.

13. After the final washing step is complete, try to drain as much TBSTT from the membrane as

possible. It is helpful to touch the corner of a dry paper towel to the edge of the membrane as

it is held at an angle. Place the membrane protein-side up in a clean tray or on plastic wrap.

14. For a typical 10 10 cm blot, 11.5 ml of the chemiluminescent substrate working solution is

sufficient. Prepare the substrate immediately before use. Wet the entire surface of the

membrane with the appropriate substrate. Incubate the blot in the substrate at room

temperature for 1 minute.

Note: CDP-Star

AP Substrate (69086-3) or SuperSignal

HRP Substrate (69059-3) are available from

Novagen for sensitive chemiluminescent detection. Use 1.5 ml of the CDP-Star

AP Substrate or

1 ml of the SupersignalHRP Substrate. Prepare the SuperSignal Substrate working solution by

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

15. Remove the membrane from the substrate. Drain any excess substrate from the membraneby touching the edge to a paper towel. Place the membrane in a Development Folder (Cat.

No. 69137-3) or on a fresh sheet of plastic wrap, and fold the plastic over the membrane.

Remove any bubbles between the plastic and the membrane. Gently remove any liquid from

the exterior of the plastic.

Optional: Place a gLOCATOR Luminescent Label (Cat. No. 69102-3) on a corner of the

Development Folder. The gLOCATOR Luminescent Label has space to record blot-

identifying data for future reference.

16. Place the wrapped membrane in a film cassette with autoradiographic film and expose for 1

10 min. An initial exposure time of 1 min is recommended. Longer exposures can be

performed although the highest light output occurs in the first five minutes. Light output

continues over several hours. Be careful not to move the film or membrane after initial

placement or multiple images can result.

Colorimetric detectionThis protocol is for the transfer of proteins and detection on nitrocellulose membranes. BSA or

gelatin is the recommended blocking reagent. In some cases, 1% Alkali-soluble Casein may be

used to reduce non-specific background but it may reduce sensitivity.

Preparation

1. Prepare 20 ml of fresh blocking solution for washing the membrane per standard 10 10 cm

blot. Use 1% BSA in 1X TBS (150 mM NaCl,10 mM Tris-HCl, pH 7.5).

2. Prepare 40 ml fresh blocking solution for diluting the primary and secondary antibodies per

standard 10 10 cm blot. Use 1% BSA in 1X TBS.

3. Prepare 80 ml 1X TBS (150 mM NaCl,10 mM Tris-HCl, pH 7.5) and 180 ml 1X TBSTT (500 mM

NaCl, 20 mM Tris-HCl, 0.05% v/v Tween-20, 0.2% v/v Triton

X-100, pH 7.5) per standard 10

10 cm blot.

4. Dilute the GSTTag Monoclonal Antibody 1:10,000. Dilute 2 l of antibody into 20 ml of

blocking solution per standard 10 10 cm blot.

5. Dilute the Goat Anti-Mouse IgG AP Conjugate (Cat. No. 69266-3) or HRP Conjugate (Cat. No.

71045-3) 1:5,000 (or as indicated by the manufacturer) in blocking solution. Dilute 4 l into 20

ml of blocking solution per standard 10 10 cm blot.

TB325 Rev.C 0503 3United States & Canada 800-207-0144Germany 0800 6931 000United Kingdom 0800 622935Or your local sales officewww.novagen.com

Novagen


4/7

GSTTagTM

Monoclonal AntibodyProtocol

The following steps should be performed at room temperature, with gentle rocking or agitation

during incubations. Use a clean tray and place the membrane protein-side up.

1. Run a SDS-polyacrylamide gel of the GSTTag fusion protein sample. Load protein markers

in an adjacent lane. Perfect Protein (Cat. No. 69959-3) or Trail Mix (Cat. No. 70982-3)

Western Markers are available from Novagen and require an S-Protein conjugate (Cat.

No.69598-3, AP, 69047-3, HRP) or HisTag Monoclonal antibody (Cat. No. 70796-3) fordetection.

2. Transfer the proteins to a membrane electrophoretically. Any standard device can be used

according to the manufacturer's instructions. The standard transfer buffer is 192 mM glycine,

25 mM Tris Base, 20% methanol, pH 8.3. If using the Perfect Protein or Trail Mix Western

markers, the 150 and 225 kDa bands may transfer incompletely due to their large size. The 15

kDa band may not efficiently bind to the membrane (particularly 0.45 pore size

nitrocellulose) due to its small size.




3. Wash the membrane twice for 5 min each time with 20 ml 1X TBS.

4. Incubate for 30 min in 20 ml blocking solution.Note: PVDF or other hydrophobic membranes may require different blocking conditions (e.g. longer

blocking times, higher concentrations of blocking reagent).

5. Wash twice for 5 min each time with 20 ml 1X TBSTT.


7. Incubate for 1 h with 20 ml GSTTag Monoclonal Antibody diluted 1:10,000 in blocking

solution.

8. Wash twice for 5 min each time with 20 ml 1X TBSTT.


10. Incubate for 1 h with 20 ml Goat Anti-Mouse IgG AP in blocking solution.

11. Wash five times for 5 min each with 20 ml 1X TBSTT. It is important to thoroughly wash the

membrane at this point to achieve maximum signal:noise ratios.

Note: Washing steps should be performed in sufficient volume and repeated 5 times to assure

complete removal of unbound conjugate. If background is evident, the blot can be washed

several more times before adding additional substrate.

12. Based on a 10 10 cm blot , prepare developing solution by combining 60 l NBT (83 mg/ml

nitro-blue tetrazolium in 70% (v/v) dimethylformamide) and 60 l BCIP (42 mg/ml 5-bromo-

4chloro-3-indoyl phosphate, toludinium salt in 100% dimethylformamide) to 15 ml of 1X AP

buffer (100 mM Tris, pH 9.5, 100 mM NaCl, 1 mM MgCl2)

The Novagen AP Detection

Reagent Kit (Cat. No.

69264-3) contains enough

NBT, BCIP and 20X AP

Buffer for 25 blots (10 cm

10 cm).

13. Place the membrane protein side up in a clean tray and add the developing solution. Incubate

the membrane at room temperature until purple color develops. Strong purple signal should

appear within 210 minutes.

14. To stop the reaction, wash the blot thoroughly in deionized water and allow to air dry. Store

dry blots at room temperature wrapped in plastic.

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GSTTagTM

Monoclonal Antibody

Immunoprecipitation Protocol

Proteins with a GSTTag sequence can be isolated from cell extracts or in vitro

transcription/translation reactions with the following immunoprecipitation protocol. The

GSTTag Monoclonal Antibody is a mouse IgG1which binds strongly to Protein G. The protocol

can be used with Protein G Plus Agarose (Oncogene Cat. No. IP08), Protein G Plus/Protein A

Agarose (Oncogene Cat. No. IP10) and MagPrep

Anti-Mouse IgG Beads (Cat. No. 70996-3).

1. Consult the manufacturers guidelines for preparation and binding capacities of the Protein G

agaroses.

2. Prepare the sample containing the GSTTag fusion protein.

a. in vitro transcription/translation reaction: Combine the desired amount of the in vitro

transcription/translation reaction with 0.5 ml 1X wash buffer (150 mM NaCl, 20 mM

Tris-HCl, 0.5% NP-40, 5 mM EDTA, 2 mM methionine, pH 8.0).

b. Cell lysates: Prepare the lysate in PBS or other buffer with a neutral pH and similar ionic

strength. The following protocol is based on harvesting approximately 25 107cells.

For further protocols to prepare cell lysates, see Ausubel et al. 1998.

Note: Inclusion of a negative control may be helpful.

3. Pre-incubate the sample with the appropriate Protein G Agarose and optionally 1 g of a

normal mouse IgG. Proteins in the sample that do not contain the GSTTag may bind non-

specifically and the pre-incubation and subsequent centrifugation will remove them.a. in vitro transcription/translation: Use 50 l of the Protein G Agarose. Vortex, incubate on

ice for 5 min and spin for 3 min.

b. Cell lysates: Use 50 l of the agarose and 5 g of a normal mouse IgG per

1 ml cell lysate sample and incubate for 40 min at 4C on a rotating shaker.

4. Centrifuge at 16,000 g for 2 min and transfer the supernatant to a new tube.

5. Add the GSTTag Antibody to the sample and incubate for 1 h at 4C on a rotating shaker.

a. in vitro transcription/translation reaction: add 1 l of the GSTTag Antibody.

b. Cell lysates: add GSTTag Monoclonal Antibody to a final concentration of 15 g/ml. The

amount of antibody can be adjusted to have an excess or an equimolar mixture of the

antibody and the GSTTag fusion protein.

6. Add the Protein G Agarose to the sample, mix well and incubate for 12 h at 4C. Calculate

the appropriate amount to add based on the manufacturers instructions.

7. Centrifuge at 16,000 g for 2 min, and remove the supernatant.

8. a. in vitro transcription/translation: Add 0.75 ml 1X wash buffer (150 mM NaCl, 20 mM Tris-

HCl, 0.5% NP-40, 5 mM EDTA, 2 mM methionine, pH 8.0).

b. Cell lysates: Add 100 l of PBS (137 mM NaCl, 4.3 mM Na2HPO

4, 2.7 mM KCl,

1.47 mM KH2PO

4, pH 7.3) and resuspend.

9. Centrifuge at 16,000 g for 2 min in a microcentrifuge and transfer supernatant to a fresh

microcentrifuge tube.

10. Repeat steps 8 and 9 twice.

11. Remove as much supernatant as possible. Resuspend the final pellet in SDS sample buffer.

a. in vitro transcription/translation: Add 50 l 2X SDS sample buffer to the pellet.

b. Cell lysates: Add 2050 l 2X SDS sample buffer.

Novagen offers 4X SDS

Sample Buffer, Cat. No.

70607-3.12. Heat for 3 min at 85C, cool to room temperature, centrifuge, and load the supernatant on a

SDS-polyacrylamide gel. Avoid the agarose when pipetting the sample.


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GSTTagTM

Monoclonal Antibody

13. Analyze the sample

a. For in vitro transcription/translation reactions that are35

S-met labeled, fix the

proteins by soaking in 10% TCA or isopropanol:water:acetic acid 25:65:10 for 20 min.

Dry the gel and expose to X-ray film. Exposure times will vary based on the amount of

radioactivity incorporated and the amount of sample used for immunoprecipitation.

Sensitivity can be increased about 10-fold using fluorography. Soak the gel in a

solution containing appropriate scintillants (e.g. Amplify-Amersham) according to the

manufacturers instructions. Use intensifying screens during exposure. A

strong signal should be observed under the following conditions:

0.22 g RNA template and 1020 Ci of > 600 Ci/mmol35

S-met in a 25 l

translation reaction

10 l of translation mix per immunoprecipitation reaction overnight (1420 h)

exposure.

b. For non-radioactively labeled samples, perform a standard Western blot. During gel

electrophoresis, the GSTTag Antibody will be released from the Protein G Agarose

Therefore during Western blotting the light and heavy chains may be

detected when using anti-mouse antibodies. Include a lane of Perfect Protein (Cat.

No. 69959-3) or Trail Mix (Cat. No. 70982-3) Western Markers to verify size.

Both of these markers can be detected with S-protein conjugates (AP Cat. No. 69598-3,

or HRP 69047-3). The HisTag

monoclonal antibody (Cat. No. 70796-3) can be used to

detect Perfect Protein Western Markers.




Immunofluorescence

We recommend the following guidelines as a starting point for your experiments. This protocol

has been verified with COS-1 and CHO-K1 cells using Cy-5 and Cy

-3 fluorophore (Amersham)

labeled secondary antibodies.

1. Prepare transfected cell cultures on coverslips or slide chambers at 1.5 105cells/cm

2.

2. Remove the medium and wash the cells with 1X PBS (137 mM NaCl, 4.3 mM Na2HPO

4, 2.7

mM KCl, 1.47 mM KH2PO4, pH 7.3) for 5 min.3. Remove the PBS and fix the cells with 4% paraformaldehyde in 1X PBS for 15 min at room

temperature.

4. Wash 2 times for 5 min each with 1X PBS at room temperature.

Note: At this point the coverslips or slide chambers may be stored for several days at 4C.

5. Permeabilize the cells with 0.3% Triton

X-100 in 1X PBS for 10 min at room temperature.

Handle the cells carefully.

6. Wash 2 times for 5 min each with 1X PBS at room temperature.

7. Block with 2% BSA and 1% Horse Serum in 1X PBS for 20 min at room temperature.

8. Incubate with a 1:10,000 dilution of GSTTag Monoclonal Antibody in 1X PBS, 0.1% Triton

X-100 and 0.2% BSA for 1 h at 37C in a humidified environment.

9. Wash 2 times for 5 min each with 1X PBS, 0.1% Triton X-100 and 0.2% BSA at room

temperature.

10. Incubate with an appropriate secondary antibody diluted in 1X PBS, 0.1% Triton X-100 and

0.2% BSA for 30 min at 37C.

11. Wash 2 times for 5 min each with 1X PBS, 0.1% Triton X-100 and 0.2% BSA at room

temperature.

12. Remove the 1X PBS and stain nuclei with 1 g/ml Hoechst 33258 (Calbiochem Cat. No.

382061) for 2 min at room temperature.

13. Wash 4 times for 5 min each with 1X PBS (no Triton X-100 or BSA) at room temperature.

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GSTTagTM

Monoclonal Antibody14. Mount the cells with mounting medium. Place one drop of mounting solution in each well or

chamber of the slide or coverslip. Cover slowly with a slide or coverslip and avoid air

bubbles. Seal the edges with nail polish and store in the dark overnight until the nail polish

has dried.

15. View under a microscope with the appropriate fluorescent filter.

Appendix APrepare a 5% Alkali-soluble Casein solution in 5X TBS from dry powder (Calbiochem Cat. No.

218680) as follows:

1. Prepare 5 M NaCl and 1 M Tris-HCl, pH 6.0.

2. Add solid casein to 70% of the final volume of deionized water and mix well. Final

concentration will be 5 g/100 ml. Allow the casein to hydrate for at least 10 min. At this point

the casein is not solubilized. An even suspension indicates complete hydration.

3. Add 10 M NaOH is small increments to solubilize the casein. As the NaOH is added, allow the

solution to equilibrate thoroughly. This process may take several hours. Avoid adding too

much NaOH or it will become very difficult to achieve the correct pH. Approximately 350 l

is needed per 100 ml.

4. When the casein is completely in solution, add the appropriate amounts of 1 M Tris (5 ml/100

ml; final concentration 50 mM) and NaCl (15 ml/100 ml; final concentration 750 mM).5. At this point the pH should be below 7.5. Adjust the pH to 7.5 with NaOH. Bring to the final

volume with deionized water.

6. Store at 4C.

References

1. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith J.A. and Struhl, K.

(1989) Current Protocols in Molecular Biology John Wiley & Sons, New York.


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TB325 GST Tag Monoclonal Antibody

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