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CBA100 20S Proteasome ELISA Kit

20S Proteasome ELISA Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
CBA100
  
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Overview

Replacement Information
Description
Overview

This product has been discontinued.





This kit is designed to measure human 20S proteasome levels in biological samples such as cell lysates and biological fluids like human plasma.
Catalogue NumberCBA100
Brand Family Calbiochem®
Application Data

Human 20S proteasome was measured as outlined in the Detailed Protocol. Absorbance was measured at dual wavelengths, 450 nm/595 nm using an ELISA plate reader (Model: Vmax kinetic microplate reader, Molecular Devices).

20S proteasome was measured as outlined in the Detailed Protocol using plasma samples from healthy donors, prepared with citrate as anticoagulant, and obtained from the San Diego Blood Bank. Samples were initially diluted 10-fold prior to carrying out the assay. Absorbance was measured at dual wavelengths, 450 nm/595 nm using an ELISA plate reader (Model: Vmax kinetic microplate reader, Molecular Devices).

Cells were cultured in DMEM with 10% FCS and harvested at 80-90% confluency. Total cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) according to the manufacturer's recommended protocol. Protein concentration in cell lysates was determined using a BCA protein assay. The concentration of 20S proteasome was measured as outlined in the Detailed Protocol. Absorbance was measured at dual wavelengths, 450 nm/595 nm using an ELISA plate reader (Model: Vmax kinetic microplate reader, Molecular Devices). The amount of 20S proteasome (in µg) in human cell lysates was calculated per mg total protein.
Materials Required but Not Delivered• Distilled H2O
• Pipettors or multi-channel pipettor carefully calibrated to the target volume

• Spectrophotometer capable of measuring absorbance at a wavelength of 450 nm with a wavelength correction of 595 nm
References
ReferencesDahlmann, B., 2007. BMC Biochem. 8 Suppl, 1:S3.
DeMartino, G.N. and Gillette, T.G. 2007.Cell 129, 659.
Jakob, C.,et al. 2007.Blood 109, 2100.
Nalepa, G.,et al. 2006.Nat. Rev. Drug Discov. 5, 596.
Product Information
Form96 tests
Format96-well plate
Applications
Biological Information
Assay range3-200 ng/ml as measured with purified, human 20S Proteasome.
Assay time3.5 hours
Sample Typecell lysates and human plasma
Physicochemical Information
Dimensions
Materials Information
Toxicological Information
Safety Information according to GHS
Safety Information
R PhraseR: 20/21/22-25-35-36/37/38-43-61

Harmful by inhalation, in contact with skin and if swallowed.
Toxic if swallowed.
Causes severe burns.
Irritating to eyes, respiratory system and skin.
May cause sensitization by skin contact.
May cause harm to the unborn child.
S PhraseS: 24/25-26-36/37/39-45-A09

Avoid contact with skin and eyes.
In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
Wear suitable protective clothing, gloves and eye/face protection.
In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
Product Usage Statements
Intended useThe Calbiochem® 20S Proteasome ELISA Kit is designed to quantitate the amount of human 20S Proteasome in biological samples, such as cell lysates and biological fluids (e.g., human plasma).
Storage and Shipping Information
Ship Code Dry Ice Only
Toxicity Multiple Toxicity Values, refer to MSDS
Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
Storage -20°C
Storage ConditionsUpon arrival store the unopened kit at -20°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:

Table 1: Storage Conditions


Note: Following initial use, the Standard, 20S Proteasome should be dispensed into aliquots and stored at -20°C. Avoid freeze/thaw cycles.
Avoid freeze/thaw Avoid freeze/thaw
Do not freeze Ok to freeze
Packaging Information
Transport Information
Supplemental Information
Specifications
Global Trade Item Number
Catalogue Number GTIN
CBA100 0

Documentation

20S Proteasome ELISA Kit Certificates of Analysis

TitleLot Number
CBA100

References

Reference overview
Dahlmann, B., 2007. BMC Biochem. 8 Suppl, 1:S3.
DeMartino, G.N. and Gillette, T.G. 2007.Cell 129, 659.
Jakob, C.,et al. 2007.Blood 109, 2100.
Nalepa, G.,et al. 2006.Nat. Rev. Drug Discov. 5, 596.
User Protocol

Revision26-October-2009 JSW
Form96 tests
Format96-well plate
StorageUpon arrival store the unopened kit at -20°C. All kit components, once opened, can be stored for up to 3 months under the following conditions:

Table 1: Storage Conditions


Note: Following initial use, the Standard, 20S Proteasome should be dispensed into aliquots and stored at -20°C. Avoid freeze/thaw cycles.
Intended useThe Calbiochem® 20S Proteasome ELISA Kit is designed to quantitate the amount of human 20S Proteasome in biological samples, such as cell lysates and biological fluids (e.g., human plasma).
Background20S proteasome, termed the core catalytic unit, together with regulatory element, PA700, form the 26S Proteasome, a 2.3 mln Da multicatalytic proteolytic particle responsible for selective degradation of polyubiquitinylated proteins. Proteasome activity is tightly controlled through the reverse process of deubiquitination. The ubiquitin-proteasome system (UPS) is considered to be a key component of cellular physiology. The 20S proteasome is a 700 Da, barrel-shaped complex composed of four rings consisting of the subunit structure, α1-7β1-7α1-7β1-7. Three subunits, β1i, β2i, and β5i, replace their constitutive counterparts under certain circumstances, resulting in further modification of the substrate specificity. The regulatory element, PA700 (also known as the19S regulatory particle) can bind to either end, or both ends, of the a rings of the 20S proteasome and serves as the gatekeeper for substrate entry. Abnormally high expression of proteasomes is found in human leukemia cells, renal cancer cells, or in breast cancer cell lines. A recent study demonstrated the significance of measuring the levels of circulating 20S proteasome in patients with multiple myeloma (MM). This study showed that an increase in 20S proteasome concentration in serum correlates with advanced disease and is an independent prognostic factor in MM.
Principles of the assayThe Calbiochem® 20S Proteasome ELISA Kit is a sandwich immunoassay that utilizes a mouse monoclonal antibody specific for the human 20S Proteasome β7 subunit, immobilized on a 96-well plate, to capture 20S proteasome from biological samples. Bound 20S proteasome is detected using a polyclonal antibody that recognizes different subunits of the 20S proteasome core complex. The amount of bound Detector Antibody is determined using a horseradish peroxidase-conjugated goat anti-rabbit polyclonal antibody. Color development is carried out using TMB chromogenic substrate. The absorbance is read using dual wavelengths of 450 nm/595 nm. The absorbance is directly proportional to the amount of 20S proteasome present in unknown samples. A highly purified, native 20S proteasome, derived from human erythrocytes, is provided as a standard.
Materials providedThe kit provides all components required to perform the assay.

• Anti-20S Proteasome Coated 96-Well Plate (Kit Component No. JA9549-1EA): 1 plate, 96-wells, supplied as twelve 8-well strips, coated with monoclonal antibody specific for human 20S proteasome
• Standard, 20S Proteasome (Kit Component No. JA9550-1UG): 1 vial, 20 µl, supplied at 50 µg/ml
• Detector Antibody (Kit Component No. JA9551-100UL): 1 vial, 100 µl, polyclonal antibody specific for 20S proteasome, supplied as 200X
• Goat Anti-Rabbit IgG HRP-Conjugate (Kit Component No. JA9552-100UL): 1 vial, 100 µl, supplied as 200X
• Detector Antibody Diluent (Kit Component No. JA9553-20ML): 1 bottle, 20 ml
• Assay Diluent (Kit Component No. JA7644-50ML): 1 bottle, 50 ml
• TMB (Kit Component No. JA1608-12ML): 1 bottle, 12 ml
• Stop Solution (Kit Component No. JA1616-12ML): 1 bottle, 12 ml
• Plate Wash Concentrate (20X) (Kit Component No. JA1617-100ML): 1 bottle, 100 ml
• Plate Sealer: 2 each
Materials Required but not provided• Distilled H2O
• Pipettors or multi-channel pipettor carefully calibrated to the target volume

• Spectrophotometer capable of measuring absorbance at a wavelength of 450 nm with a wavelength correction of 595 nm
PreparationDilute samples with Assay Diluent as needed. Example dilutions: • Normal human plasma: dilute10-fold as a starting point. • Human cell lysates (e.g., HeLa, HL-60, K 562, Hep G2, U 937) at ≥5 mg/ml total protein concentration: dilute 1000-fold as a starting point
Reagent preparationWarm all reagents to room temperature (15-25°C) just prior to use. • Standard, 20S Proteasome: Dilute Standard, 20S Protease stock solution (as provided) with Assay Diluent according to the table below. Important: Following initial thaw, the Standard, 20S Proteasome should be dispensed into aliquots and stored at -20°C. Avoid freeze/thaw cycles.

Table 2: Standard Dilutions

• 1X Detector Antibody: Dilute Detector Antibody 200-fold with Detector Antibody Diluent. To prepare enough 1X Detector Antibody to cover the entire plate, add 60 µl Detector Antibody to 11.94 ml Detector Antibody Diluent. • 1X Goat Anti-Rabbit IgG HRP-Conjugate: Dilute Goat Anti-Rabbit IgG HRP-Conjugate 200-fold with Assay Diluent. To prepare enough 1X Goat Anti-Rabbit IgG HRP-Conjugate to cover the entire plate, add 60 µl Goat Anti-Rabbit IgG HRP-Conjugate to 11.94 ml Assay Diluent • 1X Plate Wash Concentrate: Dilute Plate Wash Concentrate (20X) 20-fold with diH2O. To prepare 1000 ml 1X Plate Wash Concentrate, add 50 ml Plate Wash Concentrate (20X) to 950 ml diH2O.
Detailed protocolIt is recommended that all samples and standards be assayed in duplicate.

1. Remove the desired number of strips from the Anti-20S Proteasome Coated 96-Well Plate, return the remaining strips to foil pouch, and close the re-sealable edge.
2. Wash the wells by adding 350 µl 1X Plate Wash Buffer to each well. Empty the contents into the sink. Repeat for a total of 2 washes. Following the final wash, gently tap the inverted plate on paper towels to remove excess 1X Plate Wash Buffer.
3. Add 100 µl each 20S Proteasome Standard dilution and samples to individual designated wells. Prepare Blank wells by adding 100 µl Assay Diluent to individual designated wells.
4. Cover the plate with a Plate Sealer and incubate for 60 min at room temperature.
5. Wash the wells as outlined in step 2 above. Repeat for a total of 4-5 washes. Following the final wash, gently tap the inverted plate on paper towels to remove excess liquid.
6. Add 100 µl 1X Detector Antibody to each well. Cover the plate with a Plate Sealer and incubate for 60 min at room temperature.
7. Wash the wells as outlined in step 2 above. Repeat for a total of 5 washes. Following the final wash, gently tap the inverted plate on paper towels to remove excess liquid.
8. Add 100 µl 1X Goat Anti-Rabbit IgG HRP-Conjugate to each well. Cover the plate with a Plate Sealer and incubate for 60 min at room temperature.
9. Wash the wells as outlined in step 2 above. Repeat for a total of 6 washes. Following the final wash, gently tap the inverted plate on paper towels to remove excess liquid.
10. Add 100 µl TMB to each well. Cover the plate tightly with a Plate Sealer and incubate for 20 min at room temperature. Protect from light.
11. Add 100 µl Stop Solution to each well. Read the absorbance at dual wavelengths, 450 nm/595 nm.
CalculationsSeveral options are available for the calculation of the concentration of 20S Proteasome in assay samples. It is recommended that the data be handled by an immunoassay software package utilizing a 4 parameter logistic curve-fitting program, 4-PL. If a software package is not available, the concentration of 20S Proteasome can be calculated manually. Construct a standard curve by plotting the mean absorbance, Ab, for each standard on the Y-axis versus the corresponding concentration of 20S Proteasome on the X-axis and draw a best-fit curve through the points on the graph. If the samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.
Standard curve

Figure 1: Standard Curve

Human 20S proteasome was measured as outlined in the Detailed Protocol. Absorbance was measured at dual wavelengths, 450 nm/595 nm using an ELISA plate reader (Model: Vmax kinetic microplate reader, Molecular Devices).
Example data

Figure 2: 20S proteasome in Human Plasma

20S proteasome was measured as outlined in the Detailed Protocol using plasma samples from healthy donors, prepared with citrate as anticoagulant, and obtained from the San Diego Blood Bank. Samples were initially diluted 10-fold prior to carrying out the assay. Absorbance was measured at dual wavelengths, 450 nm/595 nm using an ELISA plate reader (Model: Vmax kinetic microplate reader, Molecular Devices).

Figure 3: 20S proteasome in Cell Lysates

Cells were cultured in DMEM with 10% FCS and harvested at 80-90% confluency. Total cell lysates were prepared using CytoBuster™ Protein Extraction Reagent (Cat. No. 71009) according to the manufacturer's recommended protocol. Protein concentration in cell lysates was determined using a BCA protein assay. The concentration of 20S proteasome was measured as outlined in the Detailed Protocol. Absorbance was measured at dual wavelengths, 450 nm/595 nm using an ELISA plate reader (Model: Vmax kinetic microplate reader, Molecular Devices). The amount of 20S proteasome (in µg) in human cell lysates was calculated per mg total protein.
Assay Range3-200 ng/ml as measured with purified, human 20S Proteasome.
Plate configuration

Table 3: Blank Plate Configuration:
Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
InteractivePathways™ is a trademark of EMD Chemicals, Inc.