Human PSMB3 (Proteasome subunit beta type-3) ELISA Kit (AEFI01102)

Product Name:	Human PSMB3 (Proteasome subunit beta type-3) ELISA Kit
SKU:	AEFI01102
Size:	96T
Reactivity:	Human
Sample Type:	Serum: 50 ul, Plasma: 50 ul, Cell Culture Supernatant: 100ul, cell or tissue lysate: 100ul, Other liquid samples: 50ul
Application:	In vitro quantitative determination of PSMB3 concentrations in serum, plasma, cell culture supernatant and other biological samples.
Detection Method:	Sandwich
Detection Duration:	4 hours
Range:	31.25-2000pg/ml
Sensitivity:	18.75pg/ml
Storage:	2-8°C(Sealed), Don't cryopreserve.
Specificity:	Specifically recognize PSMB3, no obvious cross reaction with other analogues.

This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Anti PSMB3 antibody was pre-coated onto the 96-well plate. The biotin conjugated anti PSMB3 antibody was used as the detection antibody. The standards and pilot samples were added to the wells subsequently. After incubation, unbound conjugates were removed by wash buffer. Then, biotinylated detection antibody was added to bind with PSMB3 conjugated on coated antibody. After washing off unbound conjugates, HRP-Streptavidin was added. After a third washing, TMB substrates were added to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. The concentration of PSMB3 in the sample was calculated by drawing a standard curve. The concentration of the target substance is proportional to the OD450 value.

Component	Size (96T)	Storage
ELISA Microplate (Dismountable)	8×12	Put the rest strips into a sealed foil bag with the desiccant. Stored for 1 month at 2-8°C; Stored for 6 months at -20°C
Lyophilized Standard	2 vials	Put the rest standards into a desiccant bag. Stored for 1 month at 2-8°C; Stored for 6 months at -20°C
Biotin-labeled Antibody (Concentrated, 100X)	120 ul	2-8°C (Avoid Direct Light)
HRP-Streptavidin Conjugate (SABC, 100X)	120 ul
TMB Substrate	10 ml
Sample Dilution Buffer	20 ml	2-8°C
Antibody Dilution Buffer	10 ml
SABC Dilution Buffer	10 ml
Stop Solution	10 ml
Wash Buffer (Concentrated, 25X)	30 ml
Plate Sealer	5 pieces
Product Description	1 copy

Other materials and equipment required:

Microplate reader (wavelength: 450nm)
37°C incubator (CO2 incubator for cell culture is not recommended.)
Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose)
Precision single (0.5-10μL, 5-50μL, 20-200μL, 200-1000μL) and multi-channel pipette with disposable tips (Calibration is required before use.)
Sterile tubes and Eppendorf tubes with disposable tips
Absorbent paper and loading slot
Deionized or distilled water

Recovery:

Add a certain amount of PSMB3 into the sample. Calculate the recovery by comparing the measured value with the expected amount of PSMB3 in the sample.

Sample Type	Recovery Range(%)	Average(%)
Serum(n=5)	94-104	99
EDTA Plasma(n=5)	86-103	95
Heparin Plasma(n=5)	87-103	94

Linearity:

Dilute the sample with a certain amount of PSMB3 at 1:2, 1:4 and 1:8 to get the recovery range.

Sample Type	1:2	1:4	1:8
Serum(n=5)	87-101%	86-98%	86-98%
EDTA Plasma(n=5)	93-104%	82-98%	80-99%
Heparin Plasma(n=5)	88-105%	84-91%	81-100%

Stability:

Perform the stability test for the sealed kit at 37°C and 2-8°C and get relevant data.

ELISA kit(n=5)	37°C for 1 month	2-8°C for 6 months
Average(%)	80	95-100

Precision(%):

Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate.
Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.

Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/ml)	64.74	257.8	1026.4	62.61	251.8	1020.9
Standard deviation	2.95	9.98	40.23	2.4	9.69	39.3
CV(%)	4.56	3.87	3.92	3.83	3.85	3.85

Step 1:	Add 100ul standard or sample into each well, seal the plate and static incubate for 90 minutes at 37°C. Washing: Wash the plate twice without immersion.
Step 2:	Add 100ul biotin-labeled antibody working solution into each well, seal the plate and static incubate for 60 minutes at 37°C. Washing:Wash the plate three times and immerse for 1min each time.
Step 3:	Add 100ul SABC working solution into each well, seal the plate and static incubate for 30 minutes at 37°C. Washing:Wash the plate five times and immerse for 1min each time.
Step 4:	Add 90ul TMB substrate solution, seal the plate and static incubate for 10-20 minutes at 37°C.
Step 5:	Add 50ul stop solution. Read at 450nm immediately and calculate.

Serum:	Place whole blood sample at room temperature for 2 hours or at 2-8°C overnight. Centrifuge for 20min at 1000xg and collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay.
Plasma:	EDTA-Na2/K2 is recommended as the anticoagulant. Centrifuge samples for 15 minutes at 1000×g 2-8°C within 30 minutes after collection. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. For other anticoagulant types and uses, please refer to the sample preparation guideline.
Tissue Sample:	Place the target tissue on the ice. Remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Then weigh for usage. Use lysate to grind tissue homogenates on the ice. The adding volume of lysate depends on the weight of the tissue. Usually, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors are recommended to add into the PBS (e.g. 1mM PMSF). Do further process using ultrasonic disruption or freeze-thaw cycles (Ice bath for cooling is required during ultrasonic disruption; Freeze-thaw cycles can be repeated twice.) to get the homogenates. Homogenates are then centrifuged for 5 minutes at 5000×g. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. Determine total protein concentration by BCA kit for further data analysis. Usually, total protein concentration for Elisa assay should be within 1-3mg/ml. Some tissue samples such as liver, kidney, pancreas which containing a higher endogenous peroxidase concentration may react with TMB substrate causing false positivity. In that case, try to use 1% H2O2 for 15min inactivation and perform the assay again. Notes: PBS buffer or the mild RIPA lysis can be used as lysates. While using RIPA lysis, make the PH=7.3. Avoid using any reagents containing NP-40 lysis buffer, Triton X-100 surfactant, or DTT due to their severe inhibition for kits’ working. We recommend using 50mM Tris+0.9%NaCL+0.1%SDS, PH7.3. You can prepare by yourself or contact us for purchasing.
Cell Culture Supernatant:	Collect the supernatant: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes, then collect clarified cell culture supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay.
Cell Lysate:	Suspension Cell Lysate: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes and collect cells. Then add pre-cooling PBS into collected cell and mix gently. Recollect cell by repeating centrifugation. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Lyse the cell on ice for 30min-1h or disrupt the cell by ultrasonic disruption. Adherent Cell Lysate: Absorb supernatant and add pre-cooling PBS to wash three times. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Scrape the adherent cell with cell scraper. Lyse the cell suspension added in the centrifuge tube on ice for 30min-1h or disrupt the cell by ultrasonic disruption. During lysate process, use the tip for pipetting or intermittently shake the centrifugal tube to completely lyse the protein. Mucilaginous product is DNA which can be disrupted by ultrasonic cell disruptor on ice. (3~5mm probe, 150-300W, 3~5 s/time, 30s intervals for 1~2s working). At the end of lysate or ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay. Notes: Read notes in tissue sample.
Other Biological Sample:	Centrifuge samples for 15 minutes at 1000×g at 2-8℃. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay.