![<b>Up-regulation and Antibody-Peptide Competition</b> Extracts of HeLa cells unstimulated (1) or stimulated with 1000 units/mL IFNα/β for 18 hours and 0.1 µM calyculin for 15 minutes (2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with the PKR [pT451] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2) the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphothreonine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate and signals were detected using the Pierce SuperSignalTM method. The data show that only the phosphopeptide corresponding to PKR [pT451] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of phosphorylation by the addition of IFNγ and calyculin in this testing system.](/media/images/0096417.jpeg)
BP7179 EIF2AK2 / PKR (pThr451) antibody
see related secondary antibodiessee all 21 EIF2AK2 / PKR products
0.1 ml / US$ 515
NOVUS BIOLOGICALS
PO Box 802 Littleton, CO 80160, USA
E-Mail: novus@novus-biologicals.com
Homepage: http://www.novus-biologicals.com
PO Box 802 Littleton, CO 80160, USA
E-Mail: novus@novus-biologicals.com
Homepage: http://www.novus-biologicals.com
Quick Overview
Rabbit anti EIF2AK2 / PKR (pThr451)
Synonyms
p68 kinase, eIF-2A protein kinase 2, MGC126524, PRKR, , Interferon-induced, double-stranded RNA-activated protein kinase, Interferon-inducible RNA-dependent protein kinase, Eukaryotic translation initiation factor 2-alpha kinase 2, eIF-2A protein kinase 2, Protein kinase RNA-activated, P1/eIF-2A protein kinase
Product review
Please read our product review about EIF2AK2 / PKR (pThr451): Antibodies to eukaryotic Initiation Factors (eIF).
Product Description
Rabbit anti EIF2AK2 / PKR (pThr451) , Presentation: Aff - Purified. Product is tested for Western blot / Immunoblot ( WB )
Properties
| Applications | Western blot / Immunoblot ( WB ) |
| Reactivity | Human ( Hu ), Mouse ( Ms ) |
| Presentation | Aff - Purified |
| Host | Rabbit |
| Catalog Number | BP7179 |
| Swiss Prot. No. | P19525 |
| Quantity | 0.1 ml |
| Price | US$ 515 |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BP7179.pdf |
Datasheet Extract
| Background | Double-stranded RNA-dependent protein kinase (PKR) is a 68 kDa protein that is induced by interferon and double-stranded RNAs (dsRNA) produced in virus-infected cells. Two dsRNA-binding domains in the N-terminus interact with dsRNA to modify the conformation of PKR and allow it to undergo autophosphorylation and activation. Once activated, PKR phosphorylates eIF2α, leading to inhibition of protein synthesis, growth suppression, and apoptosis induction. The phosphorylation of two sites in the activation loop of the kinase domain, threonines 446 and 451, is critical for high level catalytic activity. |
| Immunogen | Chemically synthesized phosphopeptide derived from the region of human PKR that contains threonine 451. Swiss Num.: P19525 |
| Format | State: Liquid Ig fraction Purification: Sequential epitope-specific chromatography. The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated PKR protein. The final product is generated by affinity chromatography using a PKR-derived peptide that is phosphorylated at threonine 451. BufferSystem: Dulbecco's phosphate buffered saline (without Mg2+ and Ca2+), pH 7.3 (+/- 0.1), 50% glycerol with 1.0 mg/mL BSA (IgG, protease free) as a carrier, containing 0.05 % sodum aziode as preservative |
| Applications | Western blot (1:1000 starting dilution).
Positive Control: HeLa cells stimulated with interferon alpha/beta (IFNα/β) and calyculin. |
| Specificity | This antibody detects PKR [pT451]. Species: Human, Mouse. |
| Storage | Store the antibody at -20 °C. Can be shipped at 2 - 8 °C.
Avoid repeated freezing and thawing. Centrifuge vial before opening. Shelf life: One year from despatch. |
| References | Pataer, A., et al. (2002) Adenoviral transfer of the melanoma differentiation-associated gene 7 (mda7) induces apoptosis of lung cancer cells via up-regulation of the double-stranded RNA-dependent protein kinase (PKR). Cancer Res. 62(8):2239-2243.
Vorburger, S.A., et al. (2002) Role for the double-stranded RNA activated protein kinase PKR in E2F-1-induced apoptosis. Oncogene 21(41):6278-6288. Horng, T., et al. (2001) TIRAP: an adapter molecule in the Toll signaling pathway. Nature Immunol. 2 (9):835-841. Peel, A.L., et al. (2001) Double-stranded RNA-dependent protein kinase, PKR, binds preferentially to Huntington's disease (HD) transcripts and is activated in HD tissue. Human Mol. Gen. 10(15):1531-1538. Zhang, F., et al. (2001) Binding of double-stranded RNA to protein kinase PKR is required for dimerization and promotes critical autophosphorylation events in the activation loop. J. Biol. Chem. 276(27):24946-24958. Patel, C.V., et al. (2000) PACT, a stress-modulated cellular activator of interferon-induced, double-stranded RNA activated protein kinase, PKR. J. Biol. Chem. 275(48):37993-37998. Cuddihy, A.R., et al. (1999) The double-stranded RNA activated protein kinase PKR physically associates with the tumor suppressor p53 protein and phosphorylates human p53 on serine 392 in vitro. Oncogene 18(17):2690-2702. Savinova, O., et al. (1999) Abnormal levels and minimal activity of the dsRNA-activated protein kinase, PKR, in breast carcinoma cells. Int. J. Biochem. Cell Biol. 31(1):175-189. |
| Protocols | Western Blotting Procedure
1. Lyse approximately 10e7 cells in 0.5 mL of ice cold Cell Lysis Buffer (formulation provided below). This buffer, a modified RIPA buffer, is suitable for recovery of most proteins, including membrane receptors, cytoskeletal-associated proteins, and soluble proteins. Other cell lysis buffer formulations, such as Laemmli sample buffer and Triton-X 100 buffer, are also compatible with this procedure. Additional optimization of the cell stimulation protocol and cell lysis procedure may be required for each specific application. 2. Remove the cellular debris by centrifuging the lysates at 14,000 x g for 10 minutes. Alternatively, lysates may be ultracentrifugedat 100,000 x g for 30 minutes for greater clarification. 3. Carefully decant the clarified cell lysates into clean tubes and determine the protein concentration using a suitable method, such as the Bradford assay. Polypropylene tubes are recommended for storing cell lysates. 4. React an aliquot of the lysate with an equal volume of 2x Laemmli Sample Buffer (125 mM Tris, pH 6.8, 10% glycerol, 10% SDS, 0.006% bromophenol blue, and 130 mM dithiothreitol [DTT]) and boil the mixture for 90 seconds at 100°C. 5. Load 10-30 µg of the cell lysate into the wells of an appropriate single percentage or gradient minigel and resolve the proteins by SDS-PAGE. 6. In preparation for the Western transfer, cut a piece of PVDF membrane slightly larger than the gel. Soak the membrane in methanol for 1 minute, then rinse with ddH2O for 5 minutes. Alternatively, nitrocellulose may be used. 7. Soak the membrane, 2 pieces of Whatman paper, and Western apparatus sponges in transfer buffer (formulation provided below) for 2 minutes. 8. Assemble the gel and membrane into the sandwich apparatus. 9. Transfer the proteins at 140 mA for 60-90 minutes at room temperature. 10. Following the transfer, rinse the membrane with Tris buffered saline for 2 minutes. 11. Block the membrane with blocking buffer (formulation provided below) for one hour at room temperature or overnight at 4°C. 12. Incubate the blocked blot with primary antibody at a 1:1000 starting dilution in Tris buffered saline supplemented with 3% Ig-free BSA and 0.1% Tween 20 overnight at 4°C or for two hours at room temperature. 13. Wash the blot with several changes of Tris buffered saline supplemented with 0.1% Tween 20. 14. Detect the antibody band using an appropriate secondary antibody, such as goat F(ab)2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab)2 anti-rabbit IgG horseradish peroxidase conjugate in conjunction with your chemiluminescence reagents and instrumentation. Cell Lysis Buffer Formulation: 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1 mM NaF 20 mM Na4P2O7 2 mM Na3VO4 0.1% SDS 0.5% sodium deoxycholate 1% Triton-X 100 10% glycerol 1 mM PMSF (made from a 0.3 M stock in DMSO) or 1 mM AEBSF (water soluble version of PMSF) 60 µg/mL aprotinin 10 µg/mL leupeptin 1 µg/mL pepstatin (alternatively, protease inhibitor cocktail such as Sigma Cat. # P2714 may be used) Transfer Buffer Formulation: 2.4 gm Tris base 14.2 gm glycine 200 mL methanol Q.S. to 1 liter, then add 1 mL 10% SDS. Cool to 4°C prior to use. Tris Buffered Saline Formulation: 20 mM Tris-HCl, pH 7.4 0.9% NaCl Blocking Buffer Formulation: 100 mL Tris buffered saline 5 gm BSA 0.1 mL Tween 20 Peptide Competition Experiment The specificity of a Phosphorylation Site Specific Antibody (PSSA) in each experimental system can be confirmed through peptide competition. In this technique, aliquots of antibody are pre-incubated with peptide containing the sequence of the phosphopeptide immunogen used to raise the PSSA and the corresponding non-phosphopeptide. Following preincubation with the peptide, each antibody preparation is then used as a probe in antibody-based detection methods, such as Western blotting, immunocytochemistry, flow cytometry, or ELISA. With a PSSA specific for the phosphorylated target protein, pre-incubation with an excess of peptide containing the sequence of the phosphopeptide immunogen will block all antigen binding sites, while pre-incubation with the corresponding non-phosphopeptide will not affect the antibody. In performing the Peptide Competition Experiment, it is important to note that the optimal dilutions of both antibody and peptide should be determined empirically for each specific application. The optimal dilution of antibody in these procedures is below saturating, as determined by previous experiments in your system. The optimal dilution of peptide used in these procedures will depend on the overall affinity or avidity of the antibody, as well as the quantity of the target antigen. A 50-150 fold molar excess of peptide to antibody is found to be effective for most peptide competition experiments. In the example presented below, the PSSA is used as a dilution of 1:1000 and the peptides are used at a concentration of 333 nM. The total volume of the phosphopeptide and nonphosphopeptide pre-incubated antibody preparations is 2 mL, sufficient for probing Western blot strips, as well as for use in other antibody-based detection methods. Under these conditions, the molar excess of peptide to antibody is > / = 50. Procedure: 1. Prepare three identical test samples, such as identical PVDF or nitrocellulose strips to which the protein of interest has been transferred. The test samples should be blocked using a blocking buffer, such as Tris buffered saline supplemented with 0.1% Tween 20, and either 5% BSA or 5% non-fat dried milk. 2. Prepare 6.5 mL of working antibody stock solution (1:1000 in this example) by adding 6.5 μL of antibody stock solution to 6.5 mL of buffer containing blocking protein, such as TBS supplemented with 0.1% Tween 20, and either 3% BSA or 3% non-fat dried milk. 3. Apportion the unused PSSA into working aliquots and store at -20°C for future use (the stock PSSA contains 50% glycerol and will not freeze at this temperature). 4. Allow the lyophilized control peptides to reach room temperature, ideally under desiccation. 5. Reconstitute each of the control peptides to a concentration of 66.7 µM with nanopure water. (i.e. for a peptide with a molecular mass of 1500, reconstitution with 1 mL water yields a solution with a concentration of 66.7 µM). 6. Apportion the unused reconstituted peptide solutions into working aliquots and store at -20°C for future use. 7. Label 3 test tubes as follows: - tube 1: water only no peptide control - tube 2: phosphopeptide - tube 3: non-phosphopeptide 8. Into each tube, pipette the following components - tube 1: 2 mL diluted PSSA solution plus 10 µL nanopure water - tube 2: 2 mL diluted PSSA solution plus 10 µL phosphopeptide - tube 3: 2 mL diluted PSSA solution plus 10 µL non-phosphopeptide 9. Incubate the three tubes for 30 minutes at room temperature with gentle rocking. During this incubation, the peptides have the chance to bind to the combining site of the antibody. 10. At the end of the incubation step, transfer the contents of each of the three tubes to clean reaction vessels containing one of the three identical test samples. For Western blotting strips: Incubate the strips with the pre-incubated antibody preparations for 1 hour at room temperature or overnight at 4°C. Wash each strip four times, five minutes each, to remove unbound antibody. Transfer each strip to a new solution containing a labeled secondary antibody [e.g., goat F(ab)2 anti-rabbit IgG alkaline phosphatase conjugate or goat F(ab)2 anti-rabbit IgG horseradish peroxidase conjugate. Remove unbound secondary antibody by thorough washing, and develop the signal using your chemiluminescent reagents and instrumentation. The signal obtained with antibody incubated with the "Water Only, No Peptide Control" (Tube 1), represents the maximum signal in the assay. This signal should be eliminated by preincubation with the "Phosphopeptide" (Tube 2), while pre-incubation with the "Non-Phosphopeptide" (Tube 3) should not impact the signal. If the "Phosphopeptide" only partially eliminates the signal, repeat the procedure using twice the volume of water or peptide solutions listed in Step 8. If partial competition is seen following pre-incubation with the "Non-Phosphopeptide", repeat the procedure using half the volumes of water or peptide solutions listed in Step 8. |
| Pictures | Up-regulation and Antibody-Peptide Competition Extracts of HeLa cells unstimulated (1) or stimulated with 1000 units/mL IFNα/β for 18 hours and 0.1 µM calyculin for 15 minutes (2-5) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was blocked with a 5% BSA-TBST buffer overnight at 4°C, then incubated with the PKR [pT451] antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 2) the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphothreonine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate and signals were detected using the Pierce SuperSignalTM method. The data show that only the phosphopeptide corresponding to PKR [pT451] blocks the antibody signal, demonstrating the specificity of the antibody. The data also show the induction of phosphorylation by the addition of IFNγ and calyculin in this testing system. |
12 Secondary Antibodies
| Catalog No. | Name / Host | Presentation | Reactivity | ||||
|---|---|---|---|---|---|---|---|
| R1364B | Rabbit IgG (H&L) | ||||||
| Goat | Biotin | 2 mg / US$ 305 | |||||
| R1364F | Rabbit IgG (H&L) | ||||||
| Goat | FITC | 2 mg / US$ 295 | |||||
| R1364T | Rabbit IgG (H&L) | ||||||
| Goat | TRITC | 2 mg / US$ 295 | |||||
| R1364TR | Rabbit IgG (H&L) | ||||||
| Goat | Texas Red | 2 mg / US$ 305 | |||||
| R1364HRP | Rabbit IgG (H&L) | ||||||
| Goat | HRP | 2 mg / US$ 305 | |||||
| R1364AP | Rabbit IgG (H&L) | ||||||
| Goat | AP | 1 mg / US$ 335 | |||||
| R1458C2 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy2 | 1 mg / US$ 455 | |||||
| R1458C3 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3 | 1 mg / US$ 455 | |||||
| R1458C35 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy3.5 | 1 mg / US$ 455 | |||||
| R1458C5 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5 | 1 mg / US$ 455 | |||||
| R1458C55 | Rabbit IgG (H&L) multi-species ads. | ||||||
| Goat | Cy5.5 | 1 mg / US$ 455 | |||||
| R1427R | Rabbit IgG (H&L) F(ab')2 Fragment multi-species ads. | ||||||
| Donkey | PE | 1 ml / US$ 465 | |||||
Click here to see all secondary antibodies for 'BP7179 EIF2AK2 / PKR (pThr451)'.