116820 | Anti-cAMP Rabbit pAb

116820
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      Overview

      Replacement Information

      Key Spec Table

      HostAntibody Type
      RbPolyclonal Antibody

      Pricing & Availability

      Catalogue NumberAvailability Packaging Qty/Pack Price Quantity
      US1116820-1ST
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          Description
          OverviewRecognizes cAMP. Does not exhibit cross-reactivity with other nucleotides.
          Catalogue Number116820
          Brand Family Calbiochem®
          References
          ReferencesGettys, T.W., et al. 1991. J. Biol. Chem. 266, 15949.
          Gettys, T.W., et al. 1990. Second Messengers and Phosphoproteins 13, 37.
          Corbin, J.D., et al. 1988. Methods Enzymol. 159, 74.
          Gettys, T.W., et al. 1987. J. Biol. Chem. 262, 333.
          Brooker, G., et al. 1979. Adv. Cyclic Nucleotide Res. 10, 1.
          Harper, J.F., and Brooker, G. 1975. J. Cyclic Nucleotide Res. 1, 207.
          Steiner, A.L., et al. 1972. J. Biol. Chem. 247, 1106.
          Product Information
          FormLiquid
          Formulation50 µl serum diluted in PBS and 200 µl cAMP standard in H₂O.
          PreservativeNone
          Applications
          Key Applications Radioimmunoassay
          Application NotesRadioimmunoassay (see comments)
          Application CommentsDilution of 50 µl antibody at 1:3000 will provide enough material to assay approximately 6,000 tubes. The sensitivity is <0.5 pg/50 µl in the acetylated cAMP RIA. The concentration of cAMP producing 50% displacement from total binding is 1.43 ± 0.06 pg. Variables associated with assay conditions will dictate the proper working dilution.

          Recommended Procedure for Cyclic AMP Radioimmunoassay:
          Introduction:
          The following procedure for the assay of cAMP is based on the general procedure of Harper and Brooker as modified by Gettys, et al. Antisera was raised against 2'-O-succinyl-adenosine 3',5'-cyclic monophosphate and 2'-O-succinyl-guanosine 3',5'-cyclic monophosphate as described by Steiner, et al. except that the immunogen was conjugated to bovine serum albumin. As such, the antiserum requires that standards and unknowns be acetylated prior to assay as described in the following acetylation protocol. The acetylation procedure is rapid and simple, and affords a sensitivity to <0.5 pg of cAMP and cGMP per 50 µl of sample.

          ASSAY REAGENTS:
          1. cAMP Stock Solution: a 5 µM stock solution of purified cyclic AMP in distilled water is provided with each lot of antiserum. The cAMP was purified by HPLC and the exact concentration was determined by measuring absorbance at 259 nm. The extinction coefficient of a 100 µM solution is 1.35. Working standards used in the assay are prepared fresh from the 5 µM stock solution before each assay.
          2. cAMP Antiserum: supplied as a 50 µl aliquot of purified IgG. This aliquot should be diluted 1:3000 with γ-globulin buffer (see section 4 below) prior to use in the assay, providing sufficient antibody for approximately 6,000 assays at 25 µl per tube. Depending upon the number of assays, it may be preferable to realiquot the original 50 µl of IgG into smaller sizes, and refreeze until needed.
          3. Stock Sodium Acetate-Acetic Acid Buffers: prepare these two stock buffers by weighing out 0.5 mole of sodium acetate, and dissolving in 500 ml of distilled water, and adjusting pH by adding 1.0 M acetic acid. Several hundred ml may be necessary to reach the lower pH. The final concentration will be 1.0 M at pH 4.75 and 6.2 respectively.
          4. γ-Globulin Buffer: prepare 100 ml of 50 mM sodium acetate buffer (pH 4.75) and dissolve 100 mg of human γ-globulin (Cohn Fractions II, III) in this buffer. This buffer will be used to prepare working solutions of the antiserum and iodinated cyclic AMP-TME. Store at 4°C.
          5. Secondary Antibody on Magnetic Beads: a reagent that is required but not supplied is goat anti-rabbit secondary antibody attached to magnetic beads (Advanced Magnetics, Cambridge, MA, Cat. No. 4300G). These beads are a convenient way to stop the assay; their use is described in step 5 of the cAMP assay procedure.
          6. 12% Polyethylene Glycol: dilute 50 ml of 1.0 M acetate buffer, pH 6.2 to 1 l, dissolve 120 g of polyethylene glycol (M.W. 8000), and store at 4°C. Used in step 5 of the cAMP assay procedure.
          7. Acetylation Reagents: acetylation reagents include triethylamine and acetic anhydride, and should be purchased at the highest available purity. These chemicals are moisture and light sensitive, so small aliquots (approximately 5 ml) can be stored in brown bottles. Prepare fresh aliquots each week.
          8. 125I-labeled Cyclic AMP-TME: this reagent may be obtained by iodinating of 2'-O-monosuccinyl adenosine 3',5'-cyclic monophosphate tyrosyl methyl ester (cAMP-TME), or may be purchased from a commercial source. A protocol for the iodination is given in the Iodination section of this procedure. Alternatively, commercially obtained labeled material can be diluted to 10-12,000 cpm/25 µl in the γ-globulin buffer.

          IODINATION OF CYCLIC AMP-TME:
          1. Compound to be iodinated: 2'-O-monosuccinyl-adenosine 3',5'-cyclic monophosphate tyrosyl methyl ester. Add 400 µl of 500 mM potassium phosphate buffer, pH 7.4, to 1 mg of cAMP-TME and store 10 µl aliquots (25 µg) of the compound at -70°C until used for iodination.
          2. Freshly prepare 50 ml of 50 mM potassium phosphate buffer, pH 7.4, by diluting 500 mM phosphate buffer.
          3. Prepare a solution of 25% acetic acid. This can be prepared and kept for several months. It will be used for stopping the iodination reaction.
          4. Dissolve 100 mg of Chloramine T in 10 ml of the freshly prepared 50 mM phosphate buffer. Just before use, dilute 20 µl of this solution to 2 ml with 50 mM phosphate buffer to give a 0.01% solution.
          5. The iodination is conducted by adding 20 µl of 50 mM phosphate buffer to 10 µl of cAMP-TME (25 µg), followed by 5 µl of Na125I (0.5 mCi). Add 20 µl of the 0.01% Chloramine T solution to the mixture and incubate for 30 seconds. Stop the reaction by adding 50 µl of 25% acetic acid.
          6. The resultant mixture is subjected to C-18 reverse phase HPLC to separate the iodinated cyclic AMP-TME from the free iodine. The HPLC column should be thoroughly equilibrated prior to injecting the iodinated sample. The second labeled peak should be retained and diluted 1:2 with γ-globulin buffer for storage. Alternatively, low pressure column-based methods can be used for purifying the iodinated cyclic AMP-TME; an example is described in the reference by Steiner, et al. Working solutions of labeled cyclic AMP-TME are prepared from this stock in γ-globulin buffer to give a final concentration of 20,000 cpm per 25 µl. It may be desirable to prepare commercially obtained label at lower counting rates for economy.

          PREPARATION OF SAMPLES AND STANDARDS:
          1. Tissue extracts for assay of cAMP can be prepared in a number of ways. One method described by Corbin, et al., and Gettys, et al. uses a phosphate buffer containing 20 mM EDTA and 1 mM IBMX (Cat. No. 410957). For assaying cAMP in cells adhering to plates, it is desirable to stop all cellular reactions and lyse the cells. Acid solutions containing 10% trichloroacetic acid (TCA) or 0.1 N HCl have proven suitable. When assaying adenylate cyclase in membrane preparations, the reaction is stopped with 25% TCA followed by partial neutralization of the extract with phosphate buffer. Other methods are also suitable, and in general, extracts will contain a large amount of cyclic AMP relative to the sensitivity of the assay. Hence, the extracts must be diluted to a concentration in the range of the standard curve. The important consideration is to make the dilutions in the same buffer used for the standards. 10 mM sodium acetate buffer, pH 4.75, may be used for dilution.
          2. The working standards are prepared fresh prior to each assay from the stock solution of 5 µM cAMP described in step 1 of the Assay Reagents section. Prepare a 20 nM standard by diluting 20 µl of the 5 µM stock cAMP with 4.98 ml of assay buffer. The remaining standards are made by serially diluting the 20 nM solution to give the final concentrations, all nM: 10.000, 5.000, 2.500, 1.250, 0.625, 0.312, 0.156, 0.078, 0.039, 0.020 and 0.000 (buffer with no cAMP). The standards should be made to a final volume of 1 ml.

          ACETYLATION OF STANDARDS AND SAMPLES:
          1. This procedure is carried out at room temperature, and may be done up to several hours prior to setting up the assay. However, it is a general practice to perform the acetylation immediately before setting up the assay.
          2. Place a 1 ml aliquot of each standard and sample into separate labeled tubes.
          3. In rapid succession, add 20 µl of triethylamine to each tube, followed by 10 µl of acetic anhydride and vortex immediately.

          CYCLIC AMP ASSAY PROCEDURE:
          1. Pipette duplicate 50 µl aliquots of each acetylated standard and sample into 12 x 75 mm glass tubes.
          2. Add 25 µl of diluted anti-cAMP to each tube, followed by 25 µl of iodinated cAMP. Gently mix by shaking the rack.
          3. In addition to the standards, the assay should include two tubes for estimation of nonspecific binding (NSB), and two tubes for estimation of total bound (TB) cAMP. The NSB tubes will contain 50 µl of the acetylated zero standard, 25 µl of γ-globulin buffer, and 25 µl of labeled cAMP. The TB tubes will contain 50 µl of the acetylated zero standard, 25 µl of diluted anti-cAMP and 25 µl of labeled cAMP.
          4. The assay tubes are covered and incubated overnight (16 hours) at 4°C.
          5. The assay is stopped the next day by adding 50 µl of the magnetic goat anti-rabbit secondary antibody to each tube, mixing, and incubating all tubes for 1 hour at 4°C. The magnetic beads settle out of solution, so it is necessary to resuspend them before adding 50 µl to each assay tube. After incubation, 1 ml of the cold 12% polyethylene glycol is added to each tube, and the tubes are vortexed and centrifuged for 20 minutes at 3000 rpm (4°C). The supernatant is aspirated, and another 1 ml of 12% polyethylene glycol is added to each pellet. This step is essential to reduce the background count. Do not mix. Centrifuge as before, aspirate the supernatant, and count the tubes.
          6. Representative results of a typical assay of cAMP standards are shown in Table. These values are used to construct a standard curve to quantitate the amount of cAMP in the unknown sample.
          7. The same procedure can be used to assay for cGMP using anti-cGMP.
          Biological Information
          Immunogen2ʹ-O-succinyl cAMP conjugated to BSA
          HostRabbit
          IsotypeIgG
          Antibody TypePolyclonal Antibody
          Physicochemical Information
          Sensitivity<0.5 pg acetylated cAMP in a 50 µl sample
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          Product Usage Statements
          Storage and Shipping Information
          Ship Code Dry Ice Only
          Toxicity Standard Handling
          Storage ≤ -70°C
          Avoid freeze/thaw Avoid freeze/thaw
          Do not freeze Ok to freeze
          Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
          Packaging Information
          Transport Information
          Supplemental Information
          Specifications

          Documentation

          Certificates of Analysis

          TitleLot Number
          116820

          References

          Reference overview
          Gettys, T.W., et al. 1991. J. Biol. Chem. 266, 15949.
          Gettys, T.W., et al. 1990. Second Messengers and Phosphoproteins 13, 37.
          Corbin, J.D., et al. 1988. Methods Enzymol. 159, 74.
          Gettys, T.W., et al. 1987. J. Biol. Chem. 262, 333.
          Brooker, G., et al. 1979. Adv. Cyclic Nucleotide Res. 10, 1.
          Harper, J.F., and Brooker, G. 1975. J. Cyclic Nucleotide Res. 1, 207.
          Steiner, A.L., et al. 1972. J. Biol. Chem. 247, 1106.
          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.

          Revision11-December-2007 JSW
          ApplicationRadioimmunoassay (see comments)
          DescriptionRabbit polyclonal antibody supplied as diluted serum. Recognizes cAMP. Supplied with a cAMP standard.
          HostRabbit
          Immunogen2ʹ-O-succinyl cAMP conjugated to BSA
          IsotypeIgG
          FormLiquid
          Formulation50 µl serum diluted in PBS and 200 µl cAMP standard in H₂O.
          PreservativeNone
          CommentsDilution of 50 µl antibody at 1:3000 will provide enough material to assay approximately 6,000 tubes. The sensitivity is <0.5 pg/50 µl in the acetylated cAMP RIA. The concentration of cAMP producing 50% displacement from total binding is 1.43 ± 0.06 pg. Variables associated with assay conditions will dictate the proper working dilution.

          Recommended Procedure for Cyclic AMP Radioimmunoassay:
          Introduction:
          The following procedure for the assay of cAMP is based on the general procedure of Harper and Brooker as modified by Gettys, et al. Antisera was raised against 2'-O-succinyl-adenosine 3',5'-cyclic monophosphate and 2'-O-succinyl-guanosine 3',5'-cyclic monophosphate as described by Steiner, et al. except that the immunogen was conjugated to bovine serum albumin. As such, the antiserum requires that standards and unknowns be acetylated prior to assay as described in the following acetylation protocol. The acetylation procedure is rapid and simple, and affords a sensitivity to <0.5 pg of cAMP and cGMP per 50 µl of sample.

          ASSAY REAGENTS:
          1. cAMP Stock Solution: a 5 µM stock solution of purified cyclic AMP in distilled water is provided with each lot of antiserum. The cAMP was purified by HPLC and the exact concentration was determined by measuring absorbance at 259 nm. The extinction coefficient of a 100 µM solution is 1.35. Working standards used in the assay are prepared fresh from the 5 µM stock solution before each assay.
          2. cAMP Antiserum: supplied as a 50 µl aliquot of purified IgG. This aliquot should be diluted 1:3000 with γ-globulin buffer (see section 4 below) prior to use in the assay, providing sufficient antibody for approximately 6,000 assays at 25 µl per tube. Depending upon the number of assays, it may be preferable to realiquot the original 50 µl of IgG into smaller sizes, and refreeze until needed.
          3. Stock Sodium Acetate-Acetic Acid Buffers: prepare these two stock buffers by weighing out 0.5 mole of sodium acetate, and dissolving in 500 ml of distilled water, and adjusting pH by adding 1.0 M acetic acid. Several hundred ml may be necessary to reach the lower pH. The final concentration will be 1.0 M at pH 4.75 and 6.2 respectively.
          4. γ-Globulin Buffer: prepare 100 ml of 50 mM sodium acetate buffer (pH 4.75) and dissolve 100 mg of human γ-globulin (Cohn Fractions II, III) in this buffer. This buffer will be used to prepare working solutions of the antiserum and iodinated cyclic AMP-TME. Store at 4°C.
          5. Secondary Antibody on Magnetic Beads: a reagent that is required but not supplied is goat anti-rabbit secondary antibody attached to magnetic beads (Advanced Magnetics, Cambridge, MA, Cat. No. 4300G). These beads are a convenient way to stop the assay; their use is described in step 5 of the cAMP assay procedure.
          6. 12% Polyethylene Glycol: dilute 50 ml of 1.0 M acetate buffer, pH 6.2 to 1 l, dissolve 120 g of polyethylene glycol (M.W. 8000), and store at 4°C. Used in step 5 of the cAMP assay procedure.
          7. Acetylation Reagents: acetylation reagents include triethylamine and acetic anhydride, and should be purchased at the highest available purity. These chemicals are moisture and light sensitive, so small aliquots (approximately 5 ml) can be stored in brown bottles. Prepare fresh aliquots each week.
          8. 125I-labeled Cyclic AMP-TME: this reagent may be obtained by iodinating of 2'-O-monosuccinyl adenosine 3',5'-cyclic monophosphate tyrosyl methyl ester (cAMP-TME), or may be purchased from a commercial source. A protocol for the iodination is given in the Iodination section of this procedure. Alternatively, commercially obtained labeled material can be diluted to 10-12,000 cpm/25 µl in the γ-globulin buffer.

          IODINATION OF CYCLIC AMP-TME:
          1. Compound to be iodinated: 2'-O-monosuccinyl-adenosine 3',5'-cyclic monophosphate tyrosyl methyl ester. Add 400 µl of 500 mM potassium phosphate buffer, pH 7.4, to 1 mg of cAMP-TME and store 10 µl aliquots (25 µg) of the compound at -70°C until used for iodination.
          2. Freshly prepare 50 ml of 50 mM potassium phosphate buffer, pH 7.4, by diluting 500 mM phosphate buffer.
          3. Prepare a solution of 25% acetic acid. This can be prepared and kept for several months. It will be used for stopping the iodination reaction.
          4. Dissolve 100 mg of Chloramine T in 10 ml of the freshly prepared 50 mM phosphate buffer. Just before use, dilute 20 µl of this solution to 2 ml with 50 mM phosphate buffer to give a 0.01% solution.
          5. The iodination is conducted by adding 20 µl of 50 mM phosphate buffer to 10 µl of cAMP-TME (25 µg), followed by 5 µl of Na125I (0.5 mCi). Add 20 µl of the 0.01% Chloramine T solution to the mixture and incubate for 30 seconds. Stop the reaction by adding 50 µl of 25% acetic acid.
          6. The resultant mixture is subjected to C-18 reverse phase HPLC to separate the iodinated cyclic AMP-TME from the free iodine. The HPLC column should be thoroughly equilibrated prior to injecting the iodinated sample. The second labeled peak should be retained and diluted 1:2 with γ-globulin buffer for storage. Alternatively, low pressure column-based methods can be used for purifying the iodinated cyclic AMP-TME; an example is described in the reference by Steiner, et al. Working solutions of labeled cyclic AMP-TME are prepared from this stock in γ-globulin buffer to give a final concentration of 20,000 cpm per 25 µl. It may be desirable to prepare commercially obtained label at lower counting rates for economy.

          PREPARATION OF SAMPLES AND STANDARDS:
          1. Tissue extracts for assay of cAMP can be prepared in a number of ways. One method described by Corbin, et al., and Gettys, et al. uses a phosphate buffer containing 20 mM EDTA and 1 mM IBMX (Cat. No. 410957). For assaying cAMP in cells adhering to plates, it is desirable to stop all cellular reactions and lyse the cells. Acid solutions containing 10% trichloroacetic acid (TCA) or 0.1 N HCl have proven suitable. When assaying adenylate cyclase in membrane preparations, the reaction is stopped with 25% TCA followed by partial neutralization of the extract with phosphate buffer. Other methods are also suitable, and in general, extracts will contain a large amount of cyclic AMP relative to the sensitivity of the assay. Hence, the extracts must be diluted to a concentration in the range of the standard curve. The important consideration is to make the dilutions in the same buffer used for the standards. 10 mM sodium acetate buffer, pH 4.75, may be used for dilution.
          2. The working standards are prepared fresh prior to each assay from the stock solution of 5 µM cAMP described in step 1 of the Assay Reagents section. Prepare a 20 nM standard by diluting 20 µl of the 5 µM stock cAMP with 4.98 ml of assay buffer. The remaining standards are made by serially diluting the 20 nM solution to give the final concentrations, all nM: 10.000, 5.000, 2.500, 1.250, 0.625, 0.312, 0.156, 0.078, 0.039, 0.020 and 0.000 (buffer with no cAMP). The standards should be made to a final volume of 1 ml.

          ACETYLATION OF STANDARDS AND SAMPLES:
          1. This procedure is carried out at room temperature, and may be done up to several hours prior to setting up the assay. However, it is a general practice to perform the acetylation immediately before setting up the assay.
          2. Place a 1 ml aliquot of each standard and sample into separate labeled tubes.
          3. In rapid succession, add 20 µl of triethylamine to each tube, followed by 10 µl of acetic anhydride and vortex immediately.

          CYCLIC AMP ASSAY PROCEDURE:
          1. Pipette duplicate 50 µl aliquots of each acetylated standard and sample into 12 x 75 mm glass tubes.
          2. Add 25 µl of diluted anti-cAMP to each tube, followed by 25 µl of iodinated cAMP. Gently mix by shaking the rack.
          3. In addition to the standards, the assay should include two tubes for estimation of nonspecific binding (NSB), and two tubes for estimation of total bound (TB) cAMP. The NSB tubes will contain 50 µl of the acetylated zero standard, 25 µl of γ-globulin buffer, and 25 µl of labeled cAMP. The TB tubes will contain 50 µl of the acetylated zero standard, 25 µl of diluted anti-cAMP and 25 µl of labeled cAMP.
          4. The assay tubes are covered and incubated overnight (16 hours) at 4°C.
          5. The assay is stopped the next day by adding 50 µl of the magnetic goat anti-rabbit secondary antibody to each tube, mixing, and incubating all tubes for 1 hour at 4°C. The magnetic beads settle out of solution, so it is necessary to resuspend them before adding 50 µl to each assay tube. After incubation, 1 ml of the cold 12% polyethylene glycol is added to each tube, and the tubes are vortexed and centrifuged for 20 minutes at 3000 rpm (4°C). The supernatant is aspirated, and another 1 ml of 12% polyethylene glycol is added to each pellet. This step is essential to reduce the background count. Do not mix. Centrifuge as before, aspirate the supernatant, and count the tubes.
          6. Representative results of a typical assay of cAMP standards are shown in Table. These values are used to construct a standard curve to quantitate the amount of cAMP in the unknown sample.
          7. The same procedure can be used to assay for cGMP using anti-cGMP.
          Storage ≤ -70°C
          Avoid freeze/thaw
          Do Not Freeze Ok to freeze
          Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
          Toxicity Standard Handling
          ReferencesGettys, T.W., et al. 1991. J. Biol. Chem. 266, 15949.
          Gettys, T.W., et al. 1990. Second Messengers and Phosphoproteins 13, 37.
          Corbin, J.D., et al. 1988. Methods Enzymol. 159, 74.
          Gettys, T.W., et al. 1987. J. Biol. Chem. 262, 333.
          Brooker, G., et al. 1979. Adv. Cyclic Nucleotide Res. 10, 1.
          Harper, J.F., and Brooker, G. 1975. J. Cyclic Nucleotide Res. 1, 207.
          Steiner, A.L., et al. 1972. J. Biol. Chem. 247, 1106.

          Instructions for Use

          Title
          116820 - Microbiology Anaerocult T