Product Name:	Human/Mouse RB (T821/826) Phosphorylation ELISA Kit (BA0253)
SKU:	BA0253
Detection Method:	Cell-based ELISA, fluorimetric (λex/em = 530/585 nm and 360/450 nm)
Sample Type:	Cultured cells (whole cells)
Species Reactivity:	Human, mouse and rat
Assay Time:	6.5 hours (hands-on time 2.5 hours)
Kit Size:	100 Assays
Equipment Required:	Microplate reader
Storage:	-20°C
Shelf Life:	About 6 months after receipt
Shipping:	Gel Pack

A fluorimetric cell-based ELISA for RB(T821/826) phosphorylation status in whole cells. Phosphorylated pRB(T821/826) is detected with a specific primary antibody and HRP-conjugated secondary antibody using a fluorogenic substrate (530/585 nm) and normalised to total protein (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 is necessary.
Accurate and high-throughput. Protein phosphorylation is normalized to total cellular protein in the same well, greatly minimizing well-to-well variations. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.

Determination of RB(T821/826) 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	Preparation: change pipette tips between additions of each reagent or sample; a multi-channel pipette is recommended, using separate reservoirs for each reagent. Prepare 1x Wash Buffer by diluting Stock Wash Buffer as directed (LKB1: 15-fold; RB and RPS6: 40-fold, reserving 6 mL 1x Wash Buffer for the Detection step). Assay samples in triplicate or more, with two blanks in triplicate: a Protein Blank (no cells) and a Sample Blank (cells with no Ab1, only Ab2).
2	A. Culture and treat cells. Seed 100 µL of 1-3×10^4 adherent cells (or 4-10×10^4 suspension cells) into each 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. Treat cells as desired.
3	Fix cells (caution: formaldehyde is toxic; use a chemical hood, gloves and eye protection). Adherent cells: prepare 4 wt% formaldehyde (1.3 mL 37% formaldehyde + 10.7 mL 1x Wash Buffer) and replace media with 100 µL per well. Suspension cells: prepare 8 wt% formaldehyde (2.6 mL 37% formaldehyde + 9.4 mL 1x Wash Buffer), centrifuge (500 × g, 15 min, 4°C), remove media and add 100 µL to the pellet. Incubate 20 min at room temperature (fixed plates may be stored sealed up to 2 weeks at 2-8°C).
4	Wash cells twice with 200 µL 1x Wash Buffer (gentle shaking, 3 min each). Prepare Quench Buffer (2.2 mL 3% H2O2 + 8.8 mL 1x Wash Buffer), add 100 µL per well and incubate 20 min at room temperature. Wash 3 times with 200 µL 1x Wash Buffer. Add 100 µL Blocking Buffer and incubate 1 hr at room temperature.
5	B. Add primary antibody (Ab1). Prepare 55 µL Ab1 Mixture per well (Ab1 in Blocking Buffer, 1:1000). Remove Blocking Buffer; 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. Wash 3 times with 200 µL 1x Wash Buffer.
6	C. Add secondary antibody (Ab2). Prepare 55 µL Ab2 Mixture per well (Ab2 in Blocking Buffer, 1:1000). Add 50 µL to all wells and incubate 90 min at room temperature with gentle shaking.
7	D. Detection. Wash 4 times with 200 µL 1x Wash Buffer. Prepare HRP Substrate immediately before use (LKB1: add 6 µL 3% H2O2 to the 6 mL HRP Substrate; RB and RPS6: mix 60 µL Dye Reagent with 6 mL 1x Wash Buffer and 6 µL 3% H2O2). Add 50 µL to each well and incubate 30 min at room temperature in the dark. Add 50 µL Protein Stain to each well and incubate a further 5 min in the dark.
8	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 phospho-target fluorescence at 530/585 nm for the Sample Blank ('No Ab1' wells, FpTarget.Blank) and Sample wells (FpTarget.Sample), and the mean protein fluorescence at 360/450 nm for the Protein Blank (FProtein.Blank) and Sample wells (FProtein.Sample). Specific fluorescence values: ∆FpTarget = FpTarget.Sample – FpTarget.Blank; ∆FProtein = FProtein.Sample – FProtein.Blank. Normalized pTarget = (∆FpTarget / ∆FProtein) / (∆FpTarget / ∆FProtein)o, where (∆FpTarget / ∆FProtein)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