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CBA027 Src ELISA Kit

Src ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
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      Overview

      Replacement Information
      Description
      Overview

      This product has been discontinued.

      We apologize for the inconvenience, but we do not currently have an alternative product.

      For related products, please refer to our complete listing of kinase ELISA kits.






      Detects and quantifies the levels of c-Src protein, independent of its phosphorylation state. c-Src is the member of homologous non-receptor tyrosine kinases family with two major phosphorylation sites on Src at Tyr426 and Tyr527. Activation of c-Src is critically involved in carcinoma cell adhesion, migration, metastasis and the enhancement of cell survival. Although this kit is designed for human cell lines, platelets and lymphocytes, it cross-reacts with mouse and rat cells.
      Catalogue NumberCBA027
      Brand Family Calbiochem®
      Application Data
      The analytical sensitivity of this assay is <1 ng/ml of Src (Total). This was determined by adding two standard deviations to the mean Abs obtained when the zero standard was assayed 30 times. The sensitivity of this ELISA was compared to immunoblotting using known quantities of Src. The data presented in Figure 1 show that the ELISA is at least as sensitive as immunoblotting. The bands shown in the immunoblotting data were developed using rabbit anti-Src, an alkaline phosphatase conjugated anti-rabbit IgG followed by chemiluminescent substrate and autoradiography.
      Materials Required but Not Delivered• Plate reader capable of measurement at or near 450 nm.
      • Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi-channel pipette is desirable for large assays.)
      • Cell Lysis Buffer (see Recommended Formulation).
      • 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
      ReferencesLaird, A.D., et al. 2003. Mol. Cancer Therap. 2, 461.
      Mestelin, T. and T. Hunter 2002. Science's STKE www.stke.org/cgi/content/full/OC_sigtrans;2002/115/pe3.
      Courtneidge, S.A. 2001. Biochem. Soc. Trans. 30, 11.
      Martin, G.S. 2001. Nat. Rev. Mol. Cell. Biol. 2, 467.
      Thomas, S.M. and J.S. Brugge 1997Annu. Rev. Cell Dev. Biol. 13, 513.
      Product Information
      Detection methodColorimetric
      Form96 Tests
      Format96-well plate
      Kit containsSrc Standard, Diluents, Detector Antibody, Secondary Antibody, Coated 96-Well Plate, Wash Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol.
      Applications
      Biological Information
      Assay range1.6 - 50 ng/ml
      Assay time4 h
      Sample TypeCell lysates
      Physicochemical Information
      Sensitivity≤1 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Intended useThe Calbiochem® Src (Total) ELISA is designed to detect and quantify the level of c-Src protein, independent of its phosphorylation state. Although performance characterization of the ELISA kits was done primarily on human cell lines, platelets, and lymphocytes, cross-reactivity of this kit with mouse and rat cells was observed. This assay is intended for the detection of Src from lysates of cells, and can be used to normalize the Src content of the samples when examining quantities of phosphorylated sites on Src using the Src Phospho-Specific (Tyr⁴¹⁸) ELISA kit (Cat. No. CBA028).
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Standard Handling
      Storage +2°C to +8°C
      Storage ConditionsUpon receipt, store the entire contents of the kit at +4°C.
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsSrc Standard, Diluents, Detector Antibody, Secondary Antibody, Coated 96-Well Plate, Wash Buffer, TMB Substrate, Stop Solution, Plate Sealers, and a user protocol.
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      CBA027 0

      Documentation

      Src ELISA Kit Certificates of Analysis

      TitleLot Number
      CBA027

      References

      Reference overview
      Laird, A.D., et al. 2003. Mol. Cancer Therap. 2, 461.
      Mestelin, T. and T. Hunter 2002. Science's STKE www.stke.org/cgi/content/full/OC_sigtrans;2002/115/pe3.
      Courtneidge, S.A. 2001. Biochem. Soc. Trans. 30, 11.
      Martin, G.S. 2001. Nat. Rev. Mol. Cell. Biol. 2, 467.
      Thomas, S.M. and J.S. Brugge 1997Annu. Rev. Cell Dev. Biol. 13, 513.

      Brochure

      Title
      Kit SourceBook - 2nd Edition EURO
      Kits SourceBook - 2nd Edition GBP
      Protein Kinase Assay and Detection Kits Brochure
      User Protocol

      Revision21-October-2008 RFH
      Form96 Tests
      Format96-well plate
      Detection methodColorimetric
      Specieshuman, mouse, rat
      StorageUpon receipt, store the entire contents of the kit at +4°C.
      Intended useThe Calbiochem® Src (Total) ELISA is designed to detect and quantify the level of c-Src protein, independent of its phosphorylation state. Although performance characterization of the ELISA kits was done primarily on human cell lines, platelets, and lymphocytes, cross-reactivity of this kit with mouse and rat cells was observed. This assay is intended for the detection of Src from lysates of cells, and can be used to normalize the Src content of the samples when examining quantities of phosphorylated sites on Src using the Src Phospho-Specific (Tyr⁴¹⁸) ELISA kit (Cat. No. CBA028).
      Backgroundc-Src (pp60c-src), the cellular homolog of the Rous sarcoma virus proteinv-Src, is the protein product of the c-src proto-oncogene with MW of 60 kDa. c-Src localizes to the cytoplasm, the plasma membrane,focal adhesions, and cadherin-containing cell junctions. The many functions of this protein include the promotion of cytoskeletal reorganization which influences cell adhesion and migration, the enhancement of cell survival, and the regulation of cell division. c-Src is the prototypical member of a family of homologous non-receptor tyrosine kinases. c-Src, along with Fyn and Yes, are ubiquitously expressed, while the expression of Lck, Lyn, Fgr, Hck, and Blk is limited to hematopoietic cells. Src family proteins are characterized by the presence of Src homology (SH) domains, designated SH1-SH4. c-Src's SH1 domain, located near its C terminus, contains the protein tyrosine kinase domain. The SH2 domain, located near the N terminus, mediates c-Src's association with phosphotyrosine-containing proteins. The SH3 domain, located near the N terminus, mediates c-Src's association with polyproline-containing proteins. The SH4 domain, located at the N terminus, contains a signal for myristoylation which allows c-Src to localize to the plasma membrane. c-Src also contains a C terminal regulatory domain, as well as a unique region located near its N terminus which allows it to interact with membrane-associated receptors. c-Src mediates signals arising from receptor tyrosine kinase ligand binding, G protein coupled receptor ligand binding, and integrin engagement. These stimuli regulate c-Src's activity through modulating its phosphorylation state, protein folding, protein:protein interactions, and subcellular localization. Phosphorylation of Tyr418 of human c-Src (corresponding to Tyr418 in chicken), contained in the activation loop of the kinase domain, is required for full activation. Phosphorylation of this residue is the result of intermolecular autophosphorylation. Phosphorylation of Tyr529, located in the C terminal regulatory domain, inhibits c-Src's activity. Phosphorylation of this residue is catalyzed by the kinase CSK (C terminal Src kinase). Tyr529 phosphorylation induces intramolecular folding in which the phosphorylated tyrosine interacts with Src's SH2 domain, holding the catalytic domain in an inactive state and preventing substrate binding. c-Src's activity can be restored either by Tyr529 dephosphorylation by RPTP-α, or through displacement by other SH2 domain-containing proteins. c-Src's inhibition through intramolecular folding involving the C terminal domain is regulated by phosphorylation of several N terminal serine residues by the kinase p34cdc, activating c-Src at the G2 to M phase of the cell cycle. Interestingly, the C terminal regulatory region is deleted in v-Src and in some c-Src mutations associated with human cancers, a feature which is associated with enhanced activation. c-Src is also regulated by the formation of an intramolecular association between its SH3 domain and a polyproline-containing region located between the SH2 and kinase domains. Substrates for c-Src are numerous and include: the focal adhesion, integrin, and cytoskeletal-associated proteins FAK, vinculin, cortactin, paxillin, tensin, ezrin, p130cas, p190RhoGAP, and p120RasGAP; the adherens junction proteins β- and γ-catenin, occludin, and connexin 43; the transcription factor, STAT3; the enzymes, PLCγ and PI3K; and the scaffold protein, Shc. c-Src is currently under investigation in cell cycle control, mitogen dependent and independent cell growth, anchorage-dependent and independent cell growth, cell differentiation, B and T cell development, autocrine regulation of growth factor production, transcriptional regulation, neurobiology, angiogenesis, and cancer studies. c-Src activation, observed in some human cancers, especially those of the breast and colon, is associated with an invasive, proliferative, motile cell phenotype. Drugs which specifically target c-Src are currently under development as potential cancer therapeutic agents.
      Principles of the assayThe Calbiochem® Src kit is a solid phase sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA). A monoclonal antibody specific for Src (regardless of phosphorylation state) has been coated onto the wells of the strips provided. Samples, including a standard containing Src, control specimens, and unknowns, are pipetted into these wells. During the first incubation, the Src antigen binds to the immobilized (capture) antibody. After washing, a rabbit antibody specific for Src is added to the wells. During the second incubation, this antibody serves as a detector by binding to the immobilized Src 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 Src present in the original specimen.
      Materials provided• Src Standard (Kit Component No. JA7992-1EA): Lyophilized. Refer to vial label for quantity and reconstitution volume. Standard is derived from human platelets, the donor of which was screened negative for HIV-1/2, HTLV-I/II and Hepatitis B and C, 2 vials
      • Standard Diluent Buffer (Kit Component No. JA7993-25ML): 1 bottle, 25 ml, contains 15 mM sodium azide
      • Src Antibody-Coated Wells (Kit Component No. JA79994-1EA): 1 plate, 96-wells
      • Rabbit Anti-Src (Detection Antibody) (Kit Component No. JA7995-11ML): 1 bottle, 11 ml, contains 15 mM sodium azide
      • Anti-Rabbit IgG-Horseradish Peroxidase (HRP) Concentrate, (100X) (Kit Component No. JA7995-125UL): 1 vial, 125 µl, contains 3.3 mM thymol
      • HRP Diluent (Kit Component No. JA7997-25ML): 1 bottle, 25 ml, contains 3.3 mM thymol
      • Wash Buffer Concentrate (25X) (Kit Component No. JA7998-100ML): 1 bottle, 100 ml
      • TMB Substrate (Kit Component No. JA7999-25ML): 1 bottle, 25 ml, Tetramethylbenzidine (TMB)
      • Stop Solution (Kit Component No. JA8115-25ML): 1 bottle, 25 ml per bottle
      • Plate Covers, Adhesive strips (Kit Component No. JA8116-1EA): 3 each
      Materials Required but not provided• Plate reader capable of measurement at or near 450 nm.
      • Calibrated adjustable precision pipettes, preferably with disposable plastic tips. (A manifold multi-channel pipette is desirable for large assays.)
      • Cell Lysis Buffer (see Recommended Formulation).
      • 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• Disposal 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 bags. 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 particulate matter is present, centrifuge or filter prior to analysis.
      • All standards, controls and samples should be run in duplicate.
      • Samples containing Src protein extracted from cells 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. SDS concentration should be less than 0.01% before adding to the plate.
      • 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 absorbances 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 Substrate is light sensitive, avoid prolonged exposure to light. Also avoid contact between TMB Substrate 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 solution. Let soak for 15 to 30 s, 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 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.
      PreparationCell Extraction 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 (e.g., Cat. No. 539134) reconstituted and diluted according to the supplied guideline. 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 Extraction Buffer should be thawed on ice. Important: add the protease inhibitor just before using. The stability of protease inhibitor supplemented Cell Extraction 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 Lysate: This protocol has been successfully applied to several cell lines of human origin. Researchers should optimize the cell lysate 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 cell pellet and expression of Src. For example, 108 Colo 201 cells grown in RPMI plus 10% FBS can be extracted in 1 ml of Cell Lysis Buffer. Under these conditions, use of 1-5 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 Src. 5. Transfer extract to microcentrifuge tubes and centrifuge at 13,000 rpm for 10 min at 4°C. 6. Aliquot the clear lysate to clean microfuge 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 Src Standard: Note: This Src standard was prepared from lysates of human platelets isolated from whole blood, and was calibrated against the mass of a human Src protein expressed in transfected insect cells. Subsequent lots of standard will be normalized to this lot of material to allow consistency of Src quantitation. 1. Reconstitute Src Standard with Standard Diluent Buffer. Refer to standard vial label for instructions. Swirl or mix gently and allow to sit for 10 min to ensure complete reconstitution. Label as 100 ng/ml Src. Use standard within 1 hour of reconstitution. 2. Add 0.25 ml of Standard Diluent Buffer to each of 6 tubes labeled 50, 25, 12.5, 6.25, 3.12 and 1.6 ng/ml Src. 3. Make serial dilutions of the standard as described in the following dilution table. Mix thoroughly between steps.

      Table 1: Dilution of Src Standard

      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.

      • Storage and Final Dilution of Anti-rabbit IgG Horseradish Peroxidase (HRP): Please Note: The Anti-rabbit IgG-HRP 100x concentrate is 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. Within 1 h of use, dilute 10 µl of this 100x concentrated solution with 1 ml of 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 25x concentrate to reach room temperature and mix to ensure that any precipitated salts have redissolved. Dilute 1 volume of the 25x Wash Buffer Concentrate with 24 volumes of 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 Working Wash Buffer. Store both the concentrate and the Working 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. 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. (Re-bag extra strips and frame. Store these in the refrigerator for future use.)
      2. Add 100 µl of the Standard Diluent Buffer to zero wells. Well(s) reserved for chromogen blank should be left empty.
      3. Add 100 µl of standards, samples or controls to the appropriate wells. Samples prepared in Cell Extraction 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 optimized for each experimental system. Tap gently on side of plate to thoroughly mix.
      4. Cover wells with 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. Pipette 100 µl of anti-Src (Detection Antibody) solution into each well except the chromogen blank(s). Tap gently on the side of the plate to mix.
      7. Cover wells with 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 wells with the 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 of TMB Substrate 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 is often determined by the plate reader used. Many plate readers have the capacity to record a maximum absorbance (abs) of 2.0. The abs values should be monitored and the substrate reaction stopped before the abs of the positive wells exceed the limits of the instrument. The abs 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 abs, stopping the assay after 20 to 25 min is suggested.
      14. Add 100 µl of 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 of TMB Substrate and Stop Solution. Read the plate within 2 h after 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 Src 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 producing signals higher than the highest standard (100 ng/ml) should be further diluted in Standard Diluent Buffer and re-analyzed, multiplying the concentration by the appropriate dilution factor.)
      Standard curve

      Table 3: Typical Data Obtained from Diluted Standard

      The following data was obtained for the various standards over the range of 0 to 100 ng/ml Src (Total).
      Limitations of the assayDo not extrapolate the standard curve beyond the 100 ng/ml standard point; the dose-response is non-linear in this region and accuracy is difficult to obtain. Dilute samples >100 ng/ml with Standard Diluent Buffer; re-analyze these and multiply results by the appropriate dilution factor.

      The influence of various lysate buffers has not been thoroughly investigated. The rate of degradation of native Src in various matrices has not been investigated. Although Src degradation in the Cell Lysis Buffer described in this protocol has not been seen to date, the possibility of this occurrence cannot be excluded.
      Sensitivity≤1 ng/ml
      Sensitivity Notes

      Figure 1: Sensitivity

      The analytical sensitivity of this assay is <1 ng/ml of Src (Total). This was determined by adding two standard deviations to the mean Abs obtained when the zero standard was assayed 30 times. The sensitivity of this ELISA was compared to immunoblotting using known quantities of Src. The data presented in Figure 1 show that the ELISA is at least as sensitive as immunoblotting. The bands shown in the immunoblotting data were developed using rabbit anti-Src, an alkaline phosphatase conjugated anti-rabbit IgG followed by chemiluminescent substrate and autoradiography.
      Assay Range1.6 - 50 ng/ml
      Precision

      Table 4: Intra-Assay Precision

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


      Table 5: Inter-Assay Precision

      Samples were assayed 36 times in multiple assays to determine precision between assays.
      RecoveryTo evaluate recovery, extraction buffer was diluted 1:10 with Standard Diluent Buffer to bring the SDS concentration to <0.01%. Src at various levels was spiked into the cell extract and percent recovery calculated over endogenous levels. On average, 101% recovery was observed.
      Parallelism

      Figure 2: Parallelism

      Natural Src from an extract of Colo 201 cells cultured in RPMI + 10% FCS was serially diluted in Standard Diluent Buffer. The optical density of each dilution was plotted against the Src standard curve. Parallelism demonstrated in Figure 2 indicates that the standard accurately reflects natural full length Src content in samples.
      Linearity

      Table 6: Linearity of Dilution

      Platelets were lysed with Cell Lysis Buffer. This lysate was diluted with Standard Diluent Buffer over the range of the assay and measured for Src (Total). Linear regression analysis of sample values versus the expected concentration yielded a correlation coefficient of 0.99.
      SpecificityThe Src (Total) ELISA is specific for measurement of total Src protein regardless of phosphorylation status. The following proteins were tested in the assay at 1 µg/ml and found no cross-reactivity: p38, p53 and Akt.

      The assay was found to have 100% cross-reactivity with Fyn, but not Lyn. Cross-reactivity with other Src family members was not evaluated.

      Figure 3: Specificity

      In order to demonstrate that the Src (Total) ELISA detects both phosphorylated and non-phosphorylated forms of the protein, the following study was done. Colo 201 cells were treated with PP1 (a specific inhibitor of Src Kinase) at 50 µM for 3 h, lysed, and assayed in parallel for both Src (Total) and Src Figure 3 shows that the amount of Src (Total) remained comparable, while the levels of phosphorylation at Tyr418 decreased with PP1. This "Total" assay is designed to allow normalization of Src content among samples to permit interpretation of results from the Src, Phospho-Specific (Tyr418) ELISA kit (Cat. N0. CBA028).


      Figure 4: Detection of Src in Various Cell Lines by Total ELISA

      In Figure 4, the expression of Src in various cell lines was detected by Src (Total) ELISA. The Src (Total) ELISA kit is specific for measurement of total Src, and consistent with immunoblotting (insert).
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