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CBA071 CREB ELISA Kit

CREB ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Detection Methods: Colorimetric
CBA071
  
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      Overview

      Replacement Information

      Key Spec Table

      Species ReactivityDetection Methods
      H, MColorimetric
      Description
      OverviewDetects and quantifies the level of CREB (cAMP-Response Element-Binding protein) independent of its phosphorylation state in human and mouse cells. CREB is a member of the leucine zipper family of DNA binding proteins. It induces the transcription of target genes in response to several stimuli such as peptide hormones and growth factors.
      Catalogue NumberCBA071
      Brand Family Calbiochem®
      Application Data
      The sensitivity of this ELISA was compared to immunoblotting using known quantities of CREB. The data presented in Figure 1 show that the sensitivity of the ELISA is ~4x greater than that of immunoblotting. The bands shown in the immunoblot data were developed using an anti-CREB antibody and, chemiluminescent detection.
      Materials Required but Not Delivered• PBS
      • Plate reader capable of measurement at or near 450 nm
      • Calibrated adjustable precision pipettes, preferably with disposable plastic tips (z manifold multi-channel pipette is desirable for large assays)
      • Cell lysis buffer (see Sample Preparation).
      • Deionized or distilled H2O.
      • Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.).
      • Graph paper: linear (Cartesian), log-log, or semi-log, as desired.
      • Glass or plastic tubes for diluting and aliquoting standard.
      • Absorbent paper towels.
      • Calibrated beakers and graduated cylinders in various sizes.
      References
      ReferencesImpey, S. and Goodman, R. H. 2001. Sci. STKE. 82, PE1.
      Seternes, O.M., et al. 1999 Mol. Endocrinol. 13, 1071.
      Haywitz, A.J., and Greenberg, M.E. 1999. Annu. Rev. Biochem. 68, 821.
      Xing, J., et al. 1998. Mol. Cell. Biol. 18, 1946.
      Maldonado, R., et al. 1996. Science 273, 611.
      Gonzales, G.A. and Montminy, M.R. 1989. Cell 59, 675.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsCREB Standard, Standard Diluent Buffer, CREB Antibody-Coated 96-Well Plate, Rabbit Anti-CREB (Total) Detector Antibody, Anti-Rabbit IgG-HRP Concentrate, HRP Diluent, Wash Buffer Concentrate, TMB, Stop Solution, Plate Covers and a user protocol.
      Positive controlHeLa, Jurkat, HT1080, and NIH3T3 cells
      Applications
      Biological Information
      Assay range0.156-10 ng/ml
      Assay time4 h
      Sample TypeCells
      Species Reactivity
      • Human
      • Mouse
      Physicochemical Information
      Sensitivity< 0.025 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 25-36/37/38

      Toxic if swallowed.
      Irritating to eyes, respiratory system and skin.
      S PhraseS: 26-36

      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing.
      Product Usage Statements
      Intended useThe Calbiochem® CREB ELISA Kit is designed to detect and quantify the level of CREB protein independent of its phosphorylation state. This assay also displays moderate cross reactivity to a related family member, CREM. This assay is intended for the detection of CREB from lysates of human and mouse cells. This kit can be used to normalize the phosphorylated CREB content of the samples when using the Calbiochem® PhosphoDetect™ CREB (pSer¹³³) ELISA Kit (Cat. No. CBA072).
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Storage +2°C to +8°C
      Storage ConditionsUpon arrival store the entire contents of the kit at 4°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsCREB Standard, Standard Diluent Buffer, CREB Antibody-Coated 96-Well Plate, Rabbit Anti-CREB (Total) Detector Antibody, Anti-Rabbit IgG-HRP Concentrate, HRP Diluent, Wash Buffer Concentrate, TMB, Stop Solution, Plate Covers and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA071 0

      Documentation

      References

      Reference overview
      Impey, S. and Goodman, R. H. 2001. Sci. STKE. 82, PE1.
      Seternes, O.M., et al. 1999 Mol. Endocrinol. 13, 1071.
      Haywitz, A.J., and Greenberg, M.E. 1999. Annu. Rev. Biochem. 68, 821.
      Xing, J., et al. 1998. Mol. Cell. Biol. 18, 1946.
      Maldonado, R., et al. 1996. Science 273, 611.
      Gonzales, G.A. and Montminy, M.R. 1989. Cell 59, 675.

      Brochure

      Title
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      User Protocol

      Revision15-October-2008 JSW
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman, mouse
      StorageUpon arrival store the entire contents of the kit at 4°C.
      Intended useThe Calbiochem® CREB ELISA Kit is designed to detect and quantify the level of CREB protein independent of its phosphorylation state. This assay also displays moderate cross reactivity to a related family member, CREM. This assay is intended for the detection of CREB from lysates of human and mouse cells. This kit can be used to normalize the phosphorylated CREB content of the samples when using the Calbiochem® PhosphoDetect™ CREB (pSer¹³³) ELISA Kit (Cat. No. CBA072).
      BackgroundCREB (cAMP Response Element Binding protein) is a 43 kDa protein that is a member of the large ATF/CREM/CREB transcriptional activator family. As with other members of this family, CREB contains a highly conserved leucine zipper dimerization domain and a basic DNA binding domain at its carboxyl terminus, and a unique amino terminus. CREB is ubiquitously expressed among mammalian species, and is highly conserved evolutionarily, with numerous invertebrate, plant, and yeast homologs. CREB activates transcription in response to stimuli that elevate cytoplasmic cAMP concentrations. The series of events leading to the cAMP-dependent activation of CREB is initiated by ligand binding to certain membrane receptors, which activate adenylyl cyclase. cAMP activates a protein kinase (PKA), which translocates to the nucleus, where it phosphorylates CREB at Ser133. This phosphorylation permits CREB to recruit CREB Binding Protein (CBP), and the CREB/CBP complex in turn stimulates gene expression by interacting directly with components of the general transcriptional machinery. In addition to fostering the formation of the CREB/CBP complex, the phosphorylation of Ser133 also enhances the binding of CREB to the specific DNA sequence TGACGTCA, known as the cAMP Response Element (CRE), a sequence common to the regulatory regions of genes under the control of cAMP including Bcl 2, BDNF, the immediate early gene egr 1, and cyclin D. In addition to stimuli that elevate cAMP levels and activate PKA, a variety of other stimuli are observed to induce CREB phosphorylation on Ser133. These include UV irradiation, cross linking of cell membrane proteins such as surface Ig and CD28, growth factors including PDGF, NGF, EGF, FGF, and HGF, phorbol esters, serum feeding, and Ca2+ flux that accompanies neuronal membrane depolarization. While PKA is considered to be the classical CREB kinase, other protein kinases are observed to directly phosphorylate CREB at Ser133, including the calcium/calmodulin dependent protein kinases CaMK IV and CaMK II, Rsk -1, -2, and -3 (activated by the upstream kinase ERK1/2), and MAPKAP K2 (activated by the upstream kinase, p38). Interestingly, phosphorylation of CREB at Ser133 is found to be necessary, but not sufficient to activate transcription in many model systems. Other events required for CRE-mediated transcriptional activation are currently being delineated. The regulation of gene expression by CREB and its role in cell growth, differentiation, and survival, as well as many areas of neuroscience, including learning and memory, regulation of mood, circadian rhythm, and drug addiction are active areas of investigation.
      Principles of the assayThe Calbiochem® CREB ELISA Kit is a solid phase sandwich Enzyme Linked Immuno Sorbent Assay (ELISA). A monoclonal antibody specific for CREB (regardless of phosphorylation state) has been coated onto the wells of the 96-well strips provided. Samples, including a standard containing CREB, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the CREB antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for CREB is added to the wells. During the second incubation, this antibody serves as a detection antibody by binding to the immobilized CREB protein captured during the first incubation. After removal of excess detection antibody, a horseradish peroxidase labeled anti rabbit IgG (anti rabbit IgG HRP) is added. This binds to the detection antibody to complete the four member sandwich. After a third incubation and washing to remove all the excess anti rabbit IgG HRP, a substrate solution is added, which is acted upon by the bound enzyme to produce color. The intensity of this colored product is directly proportional to the concentration of CREB present in the original specimen.
      Materials provided• CREB Standard (Kit Component Cat. No. JA9303-1EA): 2 vials, please refer to vial label for reconstitution volume
      • Standard Diluent Buffer (Kit Component Cat. No. JA9304-25ML): 1 bottle, 25 ml, contains 15 mM sodium azide
      • CREB Antibody-Coated 96-Well Plate (Kit Component Cat. No. JA9305-1EA): 1 plate, 96 wells per plate, supplied as twelve 8-well strips
      • Anti-Rabbit IgG-HRP Concentrate (Kit Component Cat. No. JA9307-125UL): 1 vial, 125 µl, supplied as 100X in 50% glycerol, contains 3.3 mM thymol
      • HRP Diluent (Kit Component Cat. No. JA9308-25ML): 1 bottle, 25 ml, contains 3.3 mM thymol
      • Wash Buffer Concentrate (Kit Component Cat. No. JA9309-100ML): 1 bottle, 100 ml, supplied as 25X
      • TMB (Kit Component Cat. No. JA9310-25ML): 1 bottle, 25 ml tetramethylbenzidine, ready-to-use
      • Stop Solution (Kit Component Cat. No. JA9311-25ML): 1 bottle, 25 ml, ready-to-use
      • Plate Cover (Kit Component Cat. No. JA9312-1EA): 3 adhesive strips
      • Rabbit Anti-CREB Detector Antibody (Kit Component Cat. No. JA9306-11ML): 1 bottle, 11 ml, contains 15 mM sodium azide
      Materials Required but not provided• PBS
      • Plate reader capable of measurement at or near 450 nm
      • Calibrated adjustable precision pipettes, preferably with disposable plastic tips (z manifold multi-channel pipette is desirable for large assays)
      • Cell lysis buffer (see Sample Preparation).
      • Deionized or distilled H2O.
      • Plate washer: automated or manual (squirt bottle, manifold dispenser, etc.).
      • Graph paper: linear (Cartesian), log-log, or semi-log, as desired.
      • Glass or plastic tubes for diluting and aliquoting standard.
      • Absorbent paper towels.
      • Calibrated beakers and graduated cylinders in various sizes.
      Precautions and recommendations• Note: This kit contains materials with small quantities of sodium azide. Sodium azide reacts with lead and copper plumbing to form explosive metal azides. Upon disposal, flush drains with a large volume of water to prevent azide accumulation. Avoid ingestion and contact with eyes, skin and mucous membranes. In case of contact, rinse affected area with plenty of water. Observe all federal, state and local regulations for disposal.
      • When not in use, kit components should be refrigerated. All reagents should be warmed to room temperature before use.
      • Plates should be allowed to come to room temperature before opening the foil bag. Once the desired number of strips has been removed, immediately reseal the bag and store at 4°C to maintain plate integrity.
      • Samples should be frozen if not analyzed shortly after collection. Avoid multiple freeze-thaw cycles of frozen samples. Thaw completely and mix well prior to analysis.
      • If large amounts of particulate matter are present, centrifuge or filter prior to analysis.
      • It is recommended that all standards, controls and samples be run in duplicate.
      • Cell lysate samples containing CREB protein should be diluted with Standard Diluent Buffer at least 1:10. This dilution is necessary to reduce the matrix effect of the cell lysis buffer.
      • When pipetting reagents, maintain a consistent order of addition from well to well. This ensures equal incubation times for all wells.
      • Cover or cap all reagents when not in use.
      • Do not mix or interchange different reagent lots from various kit lots.
      • Read absorbance within 2 h of assay completion.
      • In-house controls should be run with every assay. If control values fall outside pre-established ranges, the accuracy of the assay is suspect.
      • All residual wash liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper. Never insert absorbent paper directly into the wells.
      • Because TMB is light sensitive, avoid prolonged exposure to light. Also avoid contact between TMB and metal, or color may develop.
      • All blood components and biological materials should be handled as potentially hazardous. Follow universal precautions as established by the Centers for Disease Control and Prevention and by the Occupational Safety and Health Administration when handling and disposing infectious agents.
      • Directions for Washing: Incomplete washing will adversely affect the test outcome. All washing must be performed with Wash Buffer provided. Washing can be performed manually as follows: completely aspirate the liquid from all wells by gently lowering an aspiration tip (aspiration device) into the bottom of each well. Take care not to scratch the inside of the well. After aspiration, fill the wells with at least 0.4 ml of Diluted Wash Buffer. Let soak for 15 to 30 s and then aspirate the liquid. Repeat as directed under Detailed Protocol. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue. Alternatively, the wash solution may be put into a squirt bottle. If a squirt bottle is used, flood the plate with Diluted Wash Buffer, completely filling all wells. After the washing procedure, the plate is inverted and tapped dry on absorbent tissue. If using an automated washer, the operating instructions for washing equipment should be carefully followed. If your automated washer allows, 30 s soak cycles should be programmed into the wash cycle.
      Preparation• 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 • 1% Triton® X-100 detergent • 10% glycerol • 0.1% SDS • 0.5% deoxycholate • 1 mM PMSF (stock is 0.3 M in DMSO) • Protease Inhibitor Cocktail Set III (Cat. No. 539134) This buffer is stable for 2-3 weeks at 4°C or for up to 6 months when aliquoted (without protease inhibitors and PMSF added) and stored at -20°C. When stored frozen, the Cell Lysis Buffer should be thawed on ice. Important: add the protease inhibitors just prior to use. The stability of protease inhibitor supplemented Cell Lysis Buffer is 24 h at 4°C. PMSF is very unstable and must be added prior to use, even if added previously. • Preparation of Cell Lysates: This protocol has been applied to several cell lines with the Cell Lysis Buffer above. Researchers should optimize the cell extraction procedures for their own applications. 1. Collect cells in PBS by centrifugation (non-adherent) or scraping from culture flasks (adherent). 2. Wash cells twice with cold PBS. 3. Remove and discard the supernatant and collect the cell pellet. (At this point the cell pellet can be frozen at -80°C and lysed at a later date). 4. Lyse the cell pellet in Cell Lysis Buffer for 30 min on ice with vortexing at 10 min intervals. The volume of Cell Lysis Buffer depends on the cell number in the cell pellet and expression level of CREB. For example, 107 HeLa cells grown in DMEM plus 10% FBS can be extracted in 1 ml of Cell Lysis Buffer. Under these conditions, use of 0.1-10 µl of the clarified cell extract diluted to a volume of 100 µl/well in Standard Diluent Buffer (See Detailed Protocol) is sufficient for the detection of CREB. 5. Transfer lysates to microcentrifuge tubes and centrifuge at 13,000 rpm for 10 min at 4°C. 6. Aliquot the clear lysate to clean microcentrifuge tubes. These samples are ready for assay. Lysates can be stored at -80°C. Avoid multiple freeze/thaw cycles.
      Reagent preparation• Reconstitution and Dilution of CREB Standard: Note: This CREB standard was prepared from purified full-length recombinant human CREB protein expressed in E. coli. 1. Reconstitute the CREB Standard with Standard Diluent Buffer. Refer to the vial label for reconstitution volume. Swirl or mix gently and then leave the vial for 10 min without mixing to ensure complete reconstitution. Label as 10 ng/ml CREB. Use standard within 1 h of reconstitution. 2. Add 0.25 ml of Standard Diluent Buffer to each of 6 tubes labeled 5, 2.5, 1.25, 0.625, 0.312, and 0.156 ng/ml of CREB. 3. Make serial dilutions of the standard as described in the following dilution table. Mix thoroughly between steps. Remaining reconstituted standard should be discarded or frozen at -80°C for further use. Standard can be frozen and thawed one time only without loss of immunoreactivity.

      Table 1: Dilution of CREB Standard

      • Storage and Final Dilution of Anti-Rabbit IgG Horseradish Peroxidase (HRP): Please Note: The Anti-Rabbit IgG-HRP Concentrate is supplied in 50% glycerol. This solution is viscous. To ensure accurate dilution, allow Anti-Rabbit IgG-HRP Concentrate to reach room temperature. Gently mix. Pipette Anti-Rabbit IgG-HRP Concentrate slowly. Remove excess concentrate solution from pipette tip by gently wiping with clean absorbent paper. 1. Add 10 µl Anti-Rabbit IgG-HRP Concentrate to 1 ml HRP Diluent for each 8-well strip used in the assay. Label as Anti-Rabbit IgG-HRP Working Solution.

      Table 2: Example of Anti-rabbit IgG-HRP Working Solution

      2. Return the unused Anti-Rabbit IgG-HRP Concentrate to the refrigerator. • Dilution of Wash Buffer: Allow the Wash Buffer Concentrate to reach room temperature and mix to ensure that any precipitated salts have re-dissolved. Dilute 1 volume Wash Buffer Concentrate with 24 volumes deionized water (e.g., 50 ml may be diluted up to 1.25 liters, 100 ml may be diluted up to 2.5 liters). Label as Diluted Wash Buffer. Store both the concentrate and the Diluted Wash Buffer in the refrigerator. The diluted buffer should be used within 14 days.
      Detailed protocolBe sure to read the Precautions and Recommendations section before carrying out the assay. Allow all reagents to reach room temperature before use. Gently mix all liquid reagents prior to use.
      Note: A standard curve must be run with each assay.
      1. Determine the number of 8-well strips needed for the assay. Insert these in the frame(s) for current use. Return any unused strips to the foil pouch, reseal, and refrigerate for future use.
      2. Add 100 µl Standard Diluent Buffer to zero wells. Well(s) reserved for chromogen blank should be left empty.
      3. Add 100 µl standards, samples or controls to the appropriate wells. Samples prepared in Cell Lysis Buffer must be diluted 1:10 or greater in Standard Diluent Buffer (for example, 10 µl sample into 90 µl buffer). While a 1:10 sample dilution has been found to be satisfactory, higher dilutions such as 1:25 or 1:50 may be optimal. The dilution chosen should be optimal for each experimental system. Tap gently on side of plate to thoroughly mix.
      4. Cover the plate with a Plate Cover and incubate for 2 h at room temperature.
      5. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Directions for Washing.
      6. Add 100 µl Rabbit Anti-CREB Detector Antibody to each well except the chromogen blank(s). Tap gently on the side of the plate to mix.
      7. Cover the plate with a Plate Cover and incubate for 1 h at room temperature.
      8. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Directions for Washing.
      9. Add 100 µl Anti-Rabbit IgG-HRP Working Solution to each well except the chromogen blank(s).
      10. Cover the plate with a Plate Cover and incubate for 30 min at room temperature.
      11. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times. See Directions for Washing.
      12. Add 100 µl TMB to each well. The liquid in the wells will begin to turn blue.
      13. Incubate for 30 min at room temperature and in the dark. Please Note: Do not cover the plate with aluminum foil or metalized mylar. The incubation time for chromogen substrate development is often determined by the plate reader used. Many plate readers have the capacity to record a maximum absorbance (Abs) of 2.0. The absorbance values should be monitored and the substrate reaction stopped before the absorbance of the positive wells exceeds the limits of the instrument. The absorbance values at 450 nm can only be read after the Stop Solution has been added to each well. If using a reader that records only to 2.0 absorbance points, stopping the assay after 20 to 25 min is suggested.
      14. Add 100 µl Stop Solution to each well. Tap side of plate gently to mix. The solution in the wells should change from blue to yellow.
      15. Read the absorbance of each well at 450 nm having blanked the plate reader against a chromogen blank composed of 100 µl each TMB and Stop Solution. Read the plate within 2 h of adding the Stop Solution.
      16. Plot on graph paper the absorbance of the standards against the standard concentration. (Optimally, the background absorbance may be subtracted from all data points, including standards, unknowns and controls, prior to plotting.) Draw the best smooth curve through these points to construct the standard curve. If using curve-fitting software, the four parameter algorithm provides the best curve fit.
      17. Read the CREB concentrations for unknown samples and controls from the standard curve plotted in step 16. Multiply value(s) obtained for sample(s) by dilution factor to correct for the dilution in step 3. (Samples still producing signals higher than the highest standard (10 ng/ml) should be further diluted in Standard Diluent Buffer and re-analyzed, multiplying the concentration found by the appropriate dilution factor.)
      Standard curve

      Table 3: Typical Data Obtained with Standard

      The above data was obtained for the various standards over the range of 0 to 200 ng/ml CREB.
      Limitations of the assayDo not extrapolate the standard curve beyond the 10 ng/ml standard point; the dose-response is non-linear in this region and accuracy is difficult to obtain. Dilute samples >10 ng/ml with Standard Diluent Buffer; re-analyze these and multiply results by the appropriate dilution factor.

      The influence of various lysis buffers has not been thoroughly investigated. The rate of degradation of native CREB in various matrices has not been investigated.
      Sensitivity< 0.025 ng/ml
      Sensitivity NotesThe analytical sensitivity of this assay is <0.025 ng/ml of human CREB. This was determined by adding two standard deviations to the mean absorbance obtained when the zero standard was assayed 30 times.

      Figure 1: Sensitivity

      The sensitivity of this ELISA was compared to immunoblotting using known quantities of CREB. The data presented in Figure 1 show that the sensitivity of the ELISA is ~4x greater than that of immunoblotting. The bands shown in the immunoblot data were developed using an anti-CREB antibody and, chemiluminescent detection.
      Assay Range0.156-10 ng/ml
      Precision

      Table 4: Intra-Assay Precision

      Samples of known CREB concentration were assayed in replicates of 16 to determine precision within an assay.


      Table 5: Inter-Assay Precision

      Samples were assayed 48 times in multiple assays to determine precision between assays.
      RecoveryTo evaluate recovery, CREB Standard was spiked at 3 different concentrations into 10% cell lysis buffer. The percent recovery was calculated as an average of 103.8%.
      Parallelism

      Figure 2: Parallelism

      Natural CREB from HeLa cell lysate was serially diluted in Standard Diluent Buffer. The absorbance of each dilution was plotted against the CREB standard curve. Parallelism was demonstrated by the figure below and indicated that the standard accurately reflects CREB content in samples.
      Linearity

      Table 6: Linearity of Dilution

      HeLa cells were grown in tissue culture medium containing 10% fetal calf serum and lysed with Cell Lysis Buffer. This lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for CREB content. Linear regression analysis of samples versus the expected concentration yielded a correlation coefficient of 0.99.
      Specificity

      Figure 3: Specificity

      The CREB ELISA Kit is specific for the measurement of total CREB protein and shows moderate cross reactivity to a related family member, CREM. To determine the specificity of this ELISA kit, cell lysates from different cell lines, each at a concentration of 100 µg/ml total protein, were analyzed. The data presented in Figure 3 show that the kit detects CREB protein in cell lysates from human HeLa, HEPG2, HT1080, and Jurkat cells, and mouse NIH3T3 cells. The levels of CREB protein detected with this ELISA kit are consistent with results obtained by immunoblot analysis (insert).
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Triton® is a registered trademark of Dow Chemical Company
      Interactive Pathways™ and PhosphoDetect™ are trademarks of EMD Chemicals, Inc.