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569388 Anti-STAT3 Rabbit pAb

569388
  
Purchase on Sigma-Aldrich

Přehled

Replacement Information

Tabulka spec. kláve

Host
Rb
Description
Overview

This product has been discontinued.



We are offering Anti-Stat3 Antibody (Cat. No. 06-596) as a possible alternative. Please read the alternative product documentation carefully and contact technical service if you need additional information.






Recognizes native and denatured STAT3.
Catalogue Number569388
Brand Family Calbiochem®
SynonymsAnti-Signal Transducer and Activator of Transcription 3
Application Data
Detection of human STAT3 by immunoblotting. Samples: Whole cell lysate from HeLa cell treated with IFN-α (lane 1) or left untreated (lane 2). Primary antibody: Anti-STAT3 Rabbit pAb (Cat. No. 569388) (1:1000). Detection: chemiluminescence.
References
ReferencesDavid, M., et al. 1995. Science 269, 1721.
Ihle, J.N. 1995. Nature 377, 591.
Wen, Z., et al. 1995. Cell 82, 241.
Darnell, J.E., Jr., et al. 1994. Science 264, 1415.
Ihle, J.N., et al. 1994. Trends Biochem. Sci. 19, 222.
Product Information
FormLiquid
FormulationIn 150 mM NaCl, 10 mM HEPES, 50% glycerol, 0.01% BSA, pH 7.5.
PreservativeNone
Quality LevelMQ100
Applications
Key Applications Immunoblotting (Western Blotting)
Immunoprecipitation
Paraffin Sections
Application NotesImmunoblotting (1:1000)
Paraffin Sections (1:100, heat pre-treatment required)
Immunoprecipitation (1:100)
Application CommentsFor paraffin sections, pre-treatment in citrate buffer, pH 6.0 is required. Detects total STAT3 (phosphorylation-state independent) levels. Recognizes both native and denatured STAT3. Variables associated with assay conditions will dictate the proper working dilution.

Recommended Protocol for Immunoblotting

Solutions and Reagents
• Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol, pH 8.5.
• SDS Sample Buffer: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromphenol blue.
• 10X TBS (Tris-buffered saline): To prepare 1 liter, 24.2 g Tris base, 80 g NaCl, adjust pH to 7.6 with HCl. Dilute 1:10 for use.
• Blocking Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% non-fat dry milk.
• Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% BSA
• Wash Buffer (TBST): 1X TBS, 0.1% Tween®-20 detergent

Blotting Membrane
Nitrocellulose or PVDF membranes may be used.

Protein Blotting
A general protocol for sample preparation using 2x106 SK-N-MC cells per well in a 6-well plate is as follows:

1. Culture cells in medium containing 0.5% FBS for 2 days. We recommend plating cells directly in 0.5% FBS media to reduce basal levels of STAT3 phosphorylation.
2. Aspirate media. Add fresh media without FBS. Culture for 2 h. Note: If cells are grown at high density, changing media before treating cells with regulator reduces basal STAT3 phosphorylation due to factors secreted by cells.
3. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
4. Aspirate media from cultures; wash cells with PBS; aspirate.
5. Lyse cells by adding 100 µl SDS Sample Buffer and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice.
6. Sonicate for 2 s to shear DNA and reduce sample viscosity.
7. Heat sample to 95-100°C for 5 min. Cool on ice.
8. Microcentrifuge for 5 min.
9. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
10. Electrotransfer to nitrocellulose membrane.

As controls, we recommend using 10 µl of SK-N-MC cell extracts.

Membrane Blocking, Gel and Antibody Incubations
1. After transfer, wash membrane with 25 ml TBS for 5 min at room temperature.
2. Incubate membrane in 25 ml Blocking Buffer for 1-3 h at room temperature or overnight at 4°C.
3. Wash 3 times for 5 min each with 15 ml TBST.
4. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml Primary Antibody Dilution Buffer with gentle agitation overnight at 4°C.
5. Wash 3 times for 5 min each with 15 ml TBST.
6. Incubate membrane with conjugated secondary antibody at the appropriate dilution in 10 ml Blocking Buffer with gentle agitation for 1 h at room temperature.
7. Wash membrane as in step 5.

Detection of Proteins
Chemiluminescence.
Biological Information
Immunogena synthetic peptide corresponding to amino acids surrounding Tyr⁷⁰⁵ of mouse STAT3
ImmunogenMouse
HostRabbit
IsotypeIgG
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
Product Usage Statements
Storage and Shipping Information
Ship Code Blue Ice Only
Toxicity Standard Handling
Storage -20°C
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Special InstructionsFollowing initial thaw, aliquot and freeze (-20°C).
Packaging Information
Transport Information
Supplemental Information
Specifications
Global Trade Item Number
Katalogové číslo GTIN
569388 0

Documentation

Anti-STAT3 Rabbit pAb Certificates of Analysis

TitleLot Number
569388

References

Přehled odkazů
David, M., et al. 1995. Science 269, 1721.
Ihle, J.N. 1995. Nature 377, 591.
Wen, Z., et al. 1995. Cell 82, 241.
Darnell, J.E., Jr., et al. 1994. Science 264, 1415.
Ihle, J.N., et al. 1994. Trends Biochem. Sci. 19, 222.
Data Sheet

Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.

Revision06-July-2010 JSW
SynonymsAnti-Signal Transducer and Activator of Transcription 3
ApplicationImmunoblotting (1:1000)
Paraffin Sections (1:100, heat pre-treatment required)
Immunoprecipitation (1:100)
Application Data
Detection of human STAT3 by immunoblotting. Samples: Whole cell lysate from HeLa cell treated with IFN-α (lane 1) or left untreated (lane 2). Primary antibody: Anti-STAT3 Rabbit pAb (Cat. No. 569388) (1:1000). Detection: chemiluminescence.
DescriptionProtein A and immunoaffinity purified rabbit polyclonal antibody. Recognizes the ~90 kDa STAT3 protein.
BackgroundSTAT3 is activated by a variety of cytokines including interleukins, CNTF and EPO. Activation of STAT3 is accompanied by tyrosine phosphorylation at Tyr705 which induces dimerization, nuclear translocation and DNA binding. Transcriptional activation also appears to be regulated by serine phosphorylation (Ser727) probably via MAP-like kinases. As phosphorylation of STAT3 at Tyr705 is essential for dimerization and DNA binding, phosphorylation at this site is an excellent marker of STAT3 activity.
HostRabbit
Immunogen speciesMouse
Immunogena synthetic peptide corresponding to amino acids surrounding Tyr⁷⁰⁵ of mouse STAT3
IsotypeIgG
Specieshuman, mouse, rat
FormLiquid
FormulationIn 150 mM NaCl, 10 mM HEPES, 50% glycerol, 0.01% BSA, pH 7.5.
PreservativeNone
CommentsFor paraffin sections, pre-treatment in citrate buffer, pH 6.0 is required. Detects total STAT3 (phosphorylation-state independent) levels. Recognizes both native and denatured STAT3. Variables associated with assay conditions will dictate the proper working dilution.

Recommended Protocol for Immunoblotting

Solutions and Reagents
• Transfer Buffer: 25 mM Tris base, 0.2 M glycine, 20% methanol, pH 8.5.
• SDS Sample Buffer: 62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromphenol blue.
• 10X TBS (Tris-buffered saline): To prepare 1 liter, 24.2 g Tris base, 80 g NaCl, adjust pH to 7.6 with HCl. Dilute 1:10 for use.
• Blocking Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% non-fat dry milk.
• Primary Antibody Dilution Buffer: 1X TBS, 0.1% Tween®-20 detergent with 5% BSA
• Wash Buffer (TBST): 1X TBS, 0.1% Tween®-20 detergent

Blotting Membrane
Nitrocellulose or PVDF membranes may be used.

Protein Blotting
A general protocol for sample preparation using 2x106 SK-N-MC cells per well in a 6-well plate is as follows:

1. Culture cells in medium containing 0.5% FBS for 2 days. We recommend plating cells directly in 0.5% FBS media to reduce basal levels of STAT3 phosphorylation.
2. Aspirate media. Add fresh media without FBS. Culture for 2 h. Note: If cells are grown at high density, changing media before treating cells with regulator reduces basal STAT3 phosphorylation due to factors secreted by cells.
3. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
4. Aspirate media from cultures; wash cells with PBS; aspirate.
5. Lyse cells by adding 100 µl SDS Sample Buffer and immediately scrape the cells off the plate and transfer the extract to a microfuge tube. Keep on ice.
6. Sonicate for 2 s to shear DNA and reduce sample viscosity.
7. Heat sample to 95-100°C for 5 min. Cool on ice.
8. Microcentrifuge for 5 min.
9. Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
10. Electrotransfer to nitrocellulose membrane.

As controls, we recommend using 10 µl of SK-N-MC cell extracts.

Membrane Blocking, Gel and Antibody Incubations
1. After transfer, wash membrane with 25 ml TBS for 5 min at room temperature.
2. Incubate membrane in 25 ml Blocking Buffer for 1-3 h at room temperature or overnight at 4°C.
3. Wash 3 times for 5 min each with 15 ml TBST.
4. Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml Primary Antibody Dilution Buffer with gentle agitation overnight at 4°C.
5. Wash 3 times for 5 min each with 15 ml TBST.
6. Incubate membrane with conjugated secondary antibody at the appropriate dilution in 10 ml Blocking Buffer with gentle agitation for 1 h at room temperature.
7. Wash membrane as in step 5.

Detection of Proteins
Chemiluminescence.
Storage Avoid freeze/thaw
-20°C
Do Not Freeze Ok to freeze
Special InstructionsFollowing initial thaw, aliquot and freeze (-20°C).
Toxicity Standard Handling
ReferencesDavid, M., et al. 1995. Science 269, 1721.
Ihle, J.N. 1995. Nature 377, 591.
Wen, Z., et al. 1995. Cell 82, 241.
Darnell, J.E., Jr., et al. 1994. Science 264, 1415.
Ihle, J.N., et al. 1994. Trends Biochem. Sci. 19, 222.