Description
Human/Mouse GSK3B (S9) Phosphorylation ELISA Kit (BA0245) (BA0245)
The Human/Mouse GSK3B (S9) Phosphorylation ELISA Kit (SKU: BA0245) provides a rapid and sensitive fluorimetric cell-based assay for measuring the phosphorylation status of Glycogen Synthase Kinase-3 beta at Serine 9 directly in cultured cells. Protein phosphorylation plays a key role in regulating protein activity, and GSK3B (EC 2.7.11.1) plays an important role in energy metabolism, neuronal cell development and body pattern formation, and is a novel therapeutic target for ischaemic stroke. The assay measures phosphorylated pGSK3B(S9) using a specific primary antibody and an HRP-conjugated secondary antibody with a fluorogenic substrate, and normalises the signal to total protein measured in the same well. It eliminates the need for cell lysate preparation and reduces total assay time from the standard 21 hours to 6.5 hours, making it well suited to studying signalling pathways and the effects of inhibitors, siRNA or activators.
| Product Name: | Human/Mouse GSK3B (S9) Phosphorylation ELISA Kit (BA0245) |
| SKU: | BA0245 |
| Detection Method: | Cell-Based ELISA (FL530/585 nm, 360/450 nm) |
| Sample Type: | ['Cells'] |
| Species Reactivity: | Human, mouse and rat |
| Assay Time: | Assay takes 6.5 hrs; hands-on time 2.5 hrs |
| Kit Size: | 100 Assays |
| Equipment Required: | Microplate reader |
| Storage: | -20°C |
| Shelf Life: | 6 months |
| Shipping: | Gel Pack |
A fluorimetric cell-based ELISA kit for the determination of GSK3B(S9) phosphorylation status in whole cells and evaluation of pathway modulation by activators, inhibitors, siRNA etc. Phosphorylated pGSK3B(S9) is measured at λex/em = 530/585 nm and normalised to total cellular protein measured at 360/450 nm in the same well.
- New and improved. Total assay time reduced from the standard 21 hours to 6.5 hours (hands-on time 2.5 hrs).
- Simple and convenient. Cells are directly cultured in 96-well plates. No cell lysis necessary.
- Accurate and high-throughput. Protein phosphorylation is normalised to total cellular protein in the same well, greatly minimising well-to-well variations, and can be readily automated for thousands of samples per day.
- Determination of GSK3B(S9) phosphorylation status in whole cells.
- Evaluation of pathway modulation by activators, inhibitors, siRNA etc.
Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
| Step | Procedure |
| 1 | Prepare 1x Wash Buffer by diluting Stock Wash Buffer 40-fold with dH2O (e.g. 10 mL Stock Wash Buffer + 390 mL dH2O). Reserve 6 mL for the Detection step. Assay samples in triplicate or more, and include a Protein Blank (no cells) and a Sample Blank (cells with no Ab1, only Ab2). |
| 2 | Culture and treat cells. Seed 100 µL of 1-3×10^4 adherent cells (or 4-10×10^4 suspension cells) per well of a black 96-well culture plate; add 100 µL cell-free media into three wells for the Protein Blank. Incubate overnight at 37°C, then treat cells as desired. |
| 3 | Fix cells (caution: formaldehyde is toxic; use a chemical hood and appropriate protection). For adherent cells, replace media with 100 µL 4% formaldehyde; for suspension cells, add 100 µL 8% formaldehyde to the pellet. Incubate 20 min at room temperature, then wash twice with 200 µL 1× Wash Buffer. |
| 4 | Quench. Add 100 µL Quench Buffer (2.2 mL 3% H2O2 + 8.8 mL 1× Wash Buffer) per well; incubate 20 min at room temperature, then wash three times with 200 µL 1× Wash Buffer. |
| 5 | Block. Add 100 µL Blocking Buffer per well; incubate 1 hr at room temperature. |
| 6 | Add Primary Antibody (Ab1). Prepare 55 µL of Ab1 Mixture per well by mixing Ab1 with Blocking Buffer at 1:1000. Add 50 µL Blocking Buffer to Sample Blank wells and 50 µL Ab1 Mixture to Sample wells; incubate 90 min at room temperature (or overnight at 2-8°C) with gentle shaking, then wash three times. |
| 7 | Add Secondary Antibody (Ab2). Prepare 55 µL Ab2 Mixture per well by mixing Ab2 with Blocking Buffer at 1:1000; add 50 µL to all wells; incubate 90 min at room temperature with gentle shaking. |
| 8 | Detection. Wash four times with 200 µL 1× Wash Buffer. Prepare HRP Substrate (60 µL Dye Reagent + 6 mL 1× Wash Buffer + 6 µL 3% H2O2), add 50 µL per well and incubate 30 min at room temperature in the dark. Add 50 µL Protein Stain per well and incubate a further 5 min in the dark. |
| 9 | Read the plate at λex/em = 530/585 nm for the phosphorylated target and at λex/em = 360/450 nm for total protein. |
Calculate the mean pTarget fluorescence at 530/585 nm for the Sample Blank (“No Ab1” wells, F_pTarget.Blank) and Sample wells (F_pTarget.Sample), and the mean protein fluorescence at 360/450 nm for the Protein Blank (F_Protein.Blank) and Sample wells (F_Protein.Sample). ΔF_pTarget = F_pTarget.Sample – F_pTarget.Blank; ΔF_Protein = F_Protein.Sample – F_Protein.Blank. Normalised pTarget = (ΔF_pTarget/ΔF_Protein) / (ΔF_pTarget/ΔF_Protein)O, where (ΔF_pTarget/ΔF_Protein)O is the control reference value (e.g. time zero in kinetic studies or untreated wells in drug potency studies).
| Component | Quantity | Storage |
| Stock Wash Buffer | 25 mL | -20°C |
| Blocking Buffer | 25 mL | -20°C |
| Protein Stain | 6 mL | -20°C |
| Dye Reagent | 120 µL | -20°C |
| Ab1 | 10 µL | -20°C |
| Ab2 (gM-HRP) | 10 µL | -20°C |
