Tb325 Gst Tag Monoclonal Antibody

TM

GST•Tag Monoclonal Antibody About the Kit GST•Tag Monoclonal Antibody

50 µg 250 µg

71097-3 71097-4

Description The GST•Tag Monoclonal Antibody is a mouse monoclonal antibody (IgG1) with affinity for the 26 kDa glutathione-S-transferase (GST) domain from S. japonicum. This highly purified antibody is superior for detecting GST•Tag fusion proteins expressed in E. coli, yeast, mammalian, and in vitro transcription/translation systems by Western blotting, immunoprecipitation or immunofluorescence. GST•Tag fusion proteins can be efficiently expressed in E. coli using Novagen’s pET-41a-c(+) or pET-42a-c(+) vectors. The 50 µg 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

2.5–5 ng: Western blot developed with chromogenic substrates <1 ng:

Western blot developed with chemiluminescent substrate

Form

Stabilized solution of antibody in 50% glycerol

Working dilution

1:10,0000 for Western blotting and immunofluorescence

Components • 50 µg or 250 µg GST•Tag Monoclonal Antibody

Storage Store the GST•Tag Monoclonal Antibody at –20°C.

®

© 2003 Novagen , a brand of EMD Biosciences, Inc., an affiliate of Merck KGaA, Darmstadt, Germany. All rights reserved. gLOCATOR, GST•Tag, His•Tag, Perfect Protein, Trail Mix, The Novagen logo and Novagen name are trademarks of EMD Biosciences, Inc. CDP-Star is a trademark of Tropix, Inc. Triton is a trademark of Rohm and Haas Co. Tween is a trademark of ICI Americas Inc. SuperSignal is a trademark of Pierce Chemical Company. Cy is a trademark of Amersham Pharmacia Biotek Inc. Novagen products are sold for research use only United States & Canada 800-207-0144 Germany 0800 6931 000 United Kingdom 0800 622935 Or your local sales office www.novagen.com

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GST•Tag Monoclonal Antibody Western Blotting Chemiluminescent detection This protocol is for the transfer and detection of proteins on nitrocellulose membranes. Alkalisoluble 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 GST•Tag Monoclonal Antibody 1:10,000. Dilute 2 µl 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 µl 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.

Note:

Detection of Trail Mix Western Markers with the His•Tag Monoclonal Antibody and Goat AntiMouse IgG HRP Conjugate (H+L) is not recommended. As an alternative, use Perfect Protein Western Markers with this conjugate. 3.

Note:

Run a SDS-polyacrylamide gel of the GST•Tag 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 His•Tag® Monoclonal Antibody (Cat. No. 70796-3) for detection.

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 µ 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 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 GST•Tag Monoclonal Antibody diluted 1:10,000 in blocking solution.

9.

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

10. Wash for 5 min with 20 ml 1X TBS. 11. Incubate for 1 h with 20 ml Goat Anti-Mouse IgG AP or HRP Conjugate in blocking solution.

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GST•Tag Monoclonal Antibody Note:

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, 1–1.5 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 Supersignal HRP 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 membrane by 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 blotidentifying 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 detection This 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 GST•Tag Monoclonal Antibody 1:10,000. Dilute 2 µl 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 µl into 20 ml of blocking solution per standard 10 × 10 cm blot.

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TB325 Rev.C 0503

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GST•Tag Monoclonal Antibody Protocol 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.

Note:

Note:

1.

Run a SDS-polyacrylamide gel of the GST•Tag 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 His•Tag® Monoclonal antibody (Cat. No. 70796-3) for detection.

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 µ pore size nitrocellulose) due to its small size.

Detection of Trail Mix Western Markers with the His•Tag Monoclonal Antibody and Goat AntiMouse IgG HRP Conjugate (H+L) is not recommended. As an alternative, use Perfect Protein Western Markers with this conjugate. 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.

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.

6.

Wash for 5 min with 20 ml 1X TBS.

7.

Incubate for 1 h with 20 ml GST•Tag Monoclonal Antibody diluted 1:10,000 in blocking solution.

8.

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

9.

Wash for 5 min with 20 ml 1X TBS.

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:

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).

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 µl NBT (83 mg/ml nitro-blue tetrazolium in 70% (v/v) dimethylformamide) and 60 µl BCIP (42 mg/ml 5-bromo4chloro-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) 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 2–10 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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TB325 Rev. C 0503

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GST•Tag Monoclonal Antibody Immunoprecipitation Protocol Proteins with a GST•Tag sequence can be isolated from cell extracts or in vitro transcription/translation reactions with the following immunoprecipitation protocol. The GST•Tag Monoclonal Antibody is a mouse IgG1 which 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).

Note:

1.

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

2.

Prepare the sample containing the GST•Tag 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 7 strength. The following protocol is based on harvesting approximately 2–5 × 10 cells. For further protocols to prepare cell lysates, see Ausubel et al. 1998.

Inclusion of a negative control may be helpful. 3.

Pre-incubate the sample with the appropriate Protein G Agarose and optionally 1 µg of a normal mouse IgG. Proteins in the sample that do not contain the GST•Tag may bind nonspecifically and the pre-incubation and subsequent centrifugation will remove them. a. in vitro transcription/translation: Use 50 µl of the Protein G Agarose. Vortex, incubate on ice for 5 min and spin for 3 min. b. Cell lysates: Use 50 µl of the agarose and 5 µg of a normal mouse IgG per 1 ml cell lysate sample and incubate for 40 min at 4°C on a rotating shaker.

4.

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

5.

Add the GST•Tag Antibody to the sample and incubate for 1 h at 4°C on a rotating shaker. a. in vitro transcription/translation reaction: add 1 µl of the GST•Tag Antibody. b. Cell lysates: add GST•Tag Monoclonal Antibody to a final concentration of 1–5 µg/ml. The amount of antibody can be adjusted to have an excess or an equimolar mixture of the antibody and the GST•Tag fusion protein.

6.

Add the Protein G Agarose to the sample, mix well and incubate for 1–2 h at 4°C. 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 TrisHCl, 0.5% NP-40, 5 mM EDTA, 2 mM methionine, pH 8.0). b. Cell lysates: Add 100 µl of PBS (137 mM NaCl, 4.3 mM Na2HPO4, 2.7 mM KCl, 1.47 mM KH2PO4, 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. Novagen offers 4X SDS Sample Buffer, Cat. No. 70607-3.

11. Remove as much supernatant as possible. Resuspend the final pellet in SDS sample buffer. a. in vitro transcription/translation: Add 50 µl 2X SDS sample buffer to the pellet. b. Cell lysates: Add 20–50 µl 2X SDS sample buffer. 12. Heat for 3 min at 85°C, 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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GST•Tag Monoclonal Antibody 13. Analyze the sample a. For in vitro transcription/translation reactions that are 35S-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 manufacturer’s instructions. Use intensifying screens during exposure. A strong signal should be observed under the following conditions: 35 • 0.2–2 µg RNA template and 10–20 µCi of > 600 Ci/mmol S-met in a 25 µl translation reaction • 10 µl of translation mix per immunoprecipitation reaction overnight (14–20 h) exposure. b. For non-radioactively labeled samples, perform a standard Western blot. During gel electrophoresis, the GST•Tag 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 His•Tag monoclonal antibody (Cat. No. 70796-3) can be used to detect Perfect Protein Western Markers. Note:

Detection of Trail Mix Western Markers with the His•Tag Monoclonal Antibody and Goat AntiMouse IgG HRP Conjugate (H+L) is not recommended. As an alternative, use Perfect Protein Western Markers with this conjugate.

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.

Note:

1.

5 2 Prepare transfected cell cultures on coverslips or slide chambers at 1.5 × 10 cells/cm .

2.

Remove the medium and wash the cells with 1X PBS (137 mM NaCl, 4.3 mM Na2HPO4, 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.

At this point the coverslips or slide chambers may be stored for several days at 4°C. 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 GST•Tag Monoclonal Antibody in 1X PBS, 0.1% Triton X-100 and 0.2% BSA for 1 h at 37°C 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 37°C. 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 µg/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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GST•Tag Monoclonal Antibody 14. 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 A Prepare 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 µl 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 4°C.

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 Rev.C 0503

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