![Peptide Competition:
Extracts prepared from 3T3-L1 adipocytes treated with LIF were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% non-fat dried milk-TBST buffer overnight at 4°C, then were incubated with eIF2α [pS52] antibody for two hours at room temperature in a 3% non-fat dried milk-
TBST buffer, following prior incubation with: no peptide (1), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphoserine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat
F(ab')2 anti-rabbit IgG HRP conjugate and bands were detected using
the Pierce SuperSignal(TM) method. The data show that only the peptide corresponding to eIF2α [pS52] blocks the antibody signal, thereby demonstrating the specificity of the antibody.](/media/images/0092805.jpeg)
BP7052 EIF2A (pSer52) antibody
see related secondary antibodiessee all 27 EIF2A 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 EIF2A (pSer52)
Synonyms
eIF-2A, eIF2-alpha, EIF2S1, , Eukaryotic translation initiation factor 2 subunit 1, Eukaryotic translation initiation factor 2 subunit alpha, CDA02, MST089, MSTP004, MSTP089
Product review
Please read our product review about EIF2A (pSer52): Antibodies to eukaryotic Initiation Factors (eIF).
Product Description
Rabbit anti EIF2A (pSer52) , Presentation: Aff - Purified. Product is tested for Western blot / Immunoblot ( WB )
Properties
| Applications | Western blot / Immunoblot ( WB ) |
| Reactivity | Human ( Hu ), Mouse ( Ms ), Yeast ( Yeast ) |
| Presentation | Aff - Purified |
| Host | Rabbit |
| Catalog Number | BP7052 |
| Swiss Prot. No. | P05198 |
| Quantity | 0.1 ml |
| Price | US$ 515 |
| Delivery | Worldwide |
| Manufacturer | Acris Antibodies GmbH |
| Datasheet | view  BP7052.pdf |
Datasheet Extract
| Background | eIF2α is a 36 kDa protein which is ubiquitously expressed in many cell types. The eIF2 protein, which is composed of three subunits (α,β and γ), is one of the key molecules in the initiation of translation. In mammalian cells, stress induces eIF2alpha to be phosphorylated at serine 52 by at least two kinases: the haem-controlled repressor (HCR) and the interferon inducible double stranded RNA-dependent protein kinase (PKR). Phosphorylation of eIF2α blocks the GDP-GTP exchange activity of eIF2β, leading to inhibition of protein synthesis, growth suppression, and apoptosis induction. The phosphorylation of eIF2α is an important regulatory process in protein synthesis. |
| Immunogen | The antiserum was produced against a chemically synthesized phosphopeptide derived from the region of human eIF2α that contains serine 52. Swiss Num.: P05198 Remarks: The immunizing region is conserved among many species including rat, pig, cow, fruit fly, and yeast. |
| 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 eIF2?. The final product is generated by affinity chromatography using an eIF2?-derived peptide that is phosphorylated at serine 52. 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 and 0.05% sodium azide as preservative |
| Applications | Western blot. For Western blotting applications, we recommend using the antibody at a 1:1000 starting dilution.
Positive control used: 3T3-L1 adipocytes stimulated with Leukemia Inhibitory Factor (LIF). Previous lots of this antibody have been used in immunostaining. |
| Specificity | Reacts with eIF2α. Species: Human, mouse. |
| Storage | For short-term storage (up to one week), 2-8°C is sufficient.
For longer, store (in aliquots) at -20°C. Avoid repeated reezing and thawing. We recommend a brief centrifugation before opening to settle vial contents. Shelf life: one year from despatch. |
| References | Brush, M.H., et al. (2003) Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1 alpha to the endoplasmic reticulum and promotes dephosphorylation of the alpha subunit of eukaryotic translation initiation factor 2. Mol. Cell. Biol. 23(4):1292-1303.
Kimball, S.R., et al. (2003) Mammalian stress granules represent sites of accumulation of stalled translation initiation complexes. Am. J. Physiol. Cell Physiol. 284(2):C273-C284 (cites the use of this antibody in immunocytochemistry). 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. Singleton, R.H., et al. (2002) Traumatically induced axotomy adjacent to the soma does not result in acute neuronal death. J. Neurosci. 22(3):791-802 (cites the use of this antibody in immunohistochemistry). Soboloff, J. and S.A. Berger (2002) Sustained ER Ca2+ depletion suppresses protein synthesis and induces activationenhanced cell death in mast cells. J. Biol. Chem. 277(16):13812-13820. 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. Zhang, P., et al. (2002) The PERK eukaryotic initiation factor 2 alpha kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas. Mol. Cell. Biol. 22(11):3864-3874. Grundmann, O., et al. (2001) Repression of GCN4 mRNA translation by nitrogen starvation in Saccharomyces cerevisiae. J. Biol. Chem. 276(28):25661-25671. Li, S. and A.E Koromilas (2001) Dominant negative function by an alternatively spliced form of the interferon-inducible protein kinase PKR. J. Biol. Chem. 276(17):13881-13890. Lu, L., et al. (2001) Translation initiation control by heme-regulated eukaryotic initiation factor 2alpha kinase in erythroid cells under cytoplasmic stresses. Mol. Cell. Biol. 21(23):7971-7980 Kumar, R., et al. (2001) Brain ischemia and reperfusion activates the eukaryotic initiation factor 2 alpha kinase, PERK. J. Neurochem. 77(5):1418-1421. 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. Kimball, S.R. (1999) Eukaryotic initiation factor eIF2. Int. J. Biochem. Cell Biol. 31(1):25-29. |
| 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. This cell lysis buffer formulation is available as a separate product which requires supplementation with protease inhibitors immediately prior to use. 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 ultracentrifuged at 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 100oC. 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. 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) overnight at 4oC. 12. Incubate the blocked blot with primary antibody at a 1:1000 starting dilution in Tris buffered saline supplemented with 1% Ig-free BSA and 0.1% Tween 20 for 2 hours at room temperature or overnight at 4°C. 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. BUFFER FORMULATIONS Cell Lysis Buffer 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 µ/mL pepstatin (alternatively, protease inhibitor cocktail such as Sigma catalog number P2714 may be used) Transfer Buffer 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 4oC prior to use. Tris Buffered Saline 20 mM Tris-HCl, pH 7.4 0.9% NaCl Blocking Buffer 100 mL Tris buffered saline 5 gm BSA 0.1 mL Tween 20 Peptide Competition Experiment Phosphorylation Site Specific Antibodies (PSSAs) have been developed to enable the specific and sensitive detection of phosphorylation of particular amino acid residues in target proteins, while circumventing the need for protein purification, phosphopeptide mapping or handling radioactivity. The specificity of a 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. If an optimal antibody dilution has not been determined in your system, please refer to the Suggested Working Dilution on the antibody Product Analysis Sheet for guidance on an appropriate starting dilution. 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 non-phosphopeptide-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: 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. 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. 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). Allow the lyophilized control peptides to reach room temperature, ideally under desiccation. Reconstitute each of the control peptides (supplied at 0.1 mg/vial) to a concentration of 66.7 µM with nanopure water. For a peptide with a molecular mass of 1500, reconstitution with 1 mL water yields a solution with a concentration of 66.7 µM. Apportion the unused reconstituted peptide solutions into working aliquots and store at -20°C for future use. Label 3 test tubes as follows: tube 1: water only no peptide control tube 2: phosphopeptide tube 3: non-phosphopeptide 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 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. 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 pre-incubation 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 | Peptide Competition:
Extracts prepared from 3T3-L1 adipocytes treated with LIF were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were blocked with a 5% non-fat dried milk-TBST buffer overnight at 4°C, then were incubated with eIF2α [pS52] antibody for two hours at room temperature in a 3% non-fat dried milk- TBST buffer, following prior incubation with: no peptide (1), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphoserine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab')2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignal(TM) method. The data show that only the peptide corresponding to eIF2α [pS52] blocks the antibody signal, thereby demonstrating the specificity of the antibody. |
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 'BP7052 EIF2A (pSer52)'.