pimago -biotin Phosphoprotein Detection

Transcription

1 pimago -biotin Phosphoprotein Detection

2 INDEX Ordering Information... 3 Introduction... 3 Protocols for on Western Blot... 4 on 96-well Plate using Avidin-Fluor... 5 on 96-well Plate using Avidin-HRP... 6 Related Products... 7 Use and Use Restrictions: The Products are sold, and deliverables of any services are provided for the purposes of the buyer s internal in vitro research, development or educational use only, not for in vivo, or any therapeutic or diagnostic use, nor for resale, or for providing services or any other commercial use of any kind, including without limitation, for any transfer in any form (including as part of a kit) to a third party; for analysis or reverse engineering of the Product or for manufacturing. Products should only be used in accordance with any safety data sheets, guidance or protocols that we issue from time to time and are available for download from our Site. Protective clothing should be used at all times when handling our Products. Safety datasheets relating to all Products are available for download from the Site or upon request. Expedeon grants no other license or rights under any intellectual property in respect of Products or services deliverables and in particular grants no license to use any Product or deliverables for any commercial purposes. Sale of Products or service deliverables by us or our authorised distributors are expressly conditional upon the customer s agreement with these restrictions, which the customer gives upon placing an order for Products or deliverables. If you wish to use any Product or deliverables for any purpose other than your own internal research as described above, you will require an additional licence from Expedeon. Please contact licensing@expedeon.com.

3 ORDERING INFORMATION PRODUCT QUANTITY CAT. NO. pimago -biotin HRP - Western Blot 10 mini-blots pimago -biotin HRP - Western Blot 40 mini-blots pimago -biotin Fuor Western Blot 10 mini-blots pimago -biotin Fluor Western Blot 40 mini-blots pimago -biotin Fluor Western Blot 10 mini-blots pimago -biotin Fluor Western Blot 40 mini-blots pimago -biotin Fluor Western Blot 10 mini-blots pimago -biotin Fluor Western Blot 40 mini-blots pimago -biotin HRP Colorimetric Microplate wells pimago -biotin HRP Colorimetric Microplate wells pimago -biotin Fluor 550 Microplate wells pimago -biotin Fluor 550 Microplate wells pimago -biotin Fluor 680 Microplate wells pimago -biotin Fluor 680 Microplate wells pimago -biotin Fluor 800 Microplate wells pimago -biotin Fluor 800 Microplate wells INTRODUCTION pimago is a universal phosphoprotein detection technology that enables sensitive and specific recognition of phosphorylated molecules. pimago is based on water-soluble, globular nanopolymers (i.e., dendrimers) that are multi-functionalized with reactive groups (e.g., Ti(IV) ions) for the highly specific recognition of phosphate groups, and with reporting groups (e.g., biotin, fluorophore) that facilitate their detection. Multiple functionalized groups per dendrimer ensure very strong binding to the phosphoproteins (resulting in high specificity) and significant signal amplification from many reporting groups (resulting in high sensitivity). Unlike phospho-antibodies, the binding is not biased by amino acid sequence, and therefore can be used for detection of any phosphorylation event on any protein site. pimago detection protocol resembles a simple Western blot procedure and can be easily incorporated by any laboratory. pimago -biotin phosphoprotein detection on membrane kits. All kits include pimago -biotin reagent, streptavidin-based detection reagent, blocking buffer, pimago buffer, washing buffer and a control phosphoprotein. ECL substrate is not included. pimago -biotin phosphoprotein detection on microplate kits. All kits include pimago -biotin reagent, streptavidin-based detection reagent, blocking buffer, pimago buffer, high-binding adsorption plates and a control phosphoproteins. HRP-based kits also include colorimetry substrate and stop solution. 3

4 Components Provided: Protocol for on Western Blot Phosphorylated protein (β-casein) in LDS sample buffer as a control (load 10μL in a well; store at 4ºC); 10x Blocking buffer (need to dilute to 1x with DI water; store 10x and 1x buffers at 4ºC); 5x pimago buffer (need to dilute to 1x with DI water; store at 4 to 25ºC); 10x Washing buffer (need to dilute to 1x with DI water; store at 4 to 25ºC); 5x IAA solution (store at 4ºC); pimago reagent (store at 4ºC); avidin-hrp, if ECL-based detection (store at 4ºC); avidin-fluor, if fluorescence-based detection (store at 4ºC). Need to prepare: 1x TBST (Tris-buffered saline with 0.1% Tween 20). PROTOCOL 1. Before running the gel, boil the samples in SDS/DTT and let them cool down to room temp. Add 5x IAA solution to a 0.5x-1x final concentration directly to the samples and incubate in the dark for 15 min (this step is optional but can improve detection specificity). Load the samples onto a gel. Expedeon recommends using our RunBlue TM range of precast gels. Load one well with 10 μl of the provided phosphoprotein as a positive control (load as it is, no need to boil). 2. Run your samples and transfer onto a membrane (RunBlue Tris-Glycine Transfer Buffer (Cat # NXB86500) provides the cleanest results). *If it is desired to do fluorescent-based detection, use a special membrane with low autofluorescence.* Important Note: In many cases, the transfer system itself might contain contaminants, increasing the nonspecific background signal. To reduce this, we strongly recommend including a second piece of membrane before the gel to bind any of these contaminants (suggested set-up: filter-membrane-gel-membrane-filter). Not necessary for nitrocellulose. 3. Block the membrane for 1hr with a 1x Blocking buffer (e.g. 10 ml for a mini blot; this step can also be carried out overnight at 4ºC). 4. Prepare 1:1,000 mixture of pimago reagent in 1x pimago buffer (e.g. 10 μl pimago in 10 ml pimago buffer for mini gel). Mix and add to the membrane, incubate 1 hour. 5. Wash the membrane 3 times with 10-20mL of 1x Wash buffer and once with 1x TBST (5 min each wash). 6. Prepare 1:1,000 mixture of avidin-hrp or avidin-fluor in the 1x Blocking buffer (e.g. 10 μl avidin reagent in 10 ml of blocking buffer for mini gel). Mix and add to the membrane, incubate 1 hour. 7. Wash the membrane 3 times with 1x TBST (5 min each wash). Detect the signal as usual using scanner or HRP chemiluminescence substrate. Expedeon recommends using our range of LumiBlue ECL solutions. (Typically, do not need to expose the film for more than 1-2 min to avoid high background; no need to dry the membrane for fluorescence detection). Note: For nitrocellulose membrane, it is sometimes observed that HRP might go through ECL substrate too fast and no signal is detected. In this case, rinse the membrane with TBST and add more ECL substrate for repeat detection. 4

5 Components Provided: Protocol for on 96-well Plate using Avidin-Fluor Phosphorylated protein (β-casein) as a control (mix 5 μl with 95 μl Binding buffer; store at 4ºC); Blocking buffer (store at 4ºC); pimago buffer (store at 4 to 25ºC); Binding buffer (store at 4ºC); pimago reagent (store at 4ºC); avidin-fluor (store at 4ºC); 96-well plate for fluorescence detection. Need to prepare: 1x TBST (Tris-buffered saline with 0.1% Tween 20). PROTOCOL 1. Binding of samples to the 96-well plate Prepare a protein solution of your sample (phosphoprotein or substrate of interest) in Binding buffer. A dephosphorylated (e.g. alkaline phosphatase-treated or without ATP) form of the sample should also be prepared as a reference. If protein amount is known, use 10 to 500 ng of the protein or mixture of proteins per 100 μl of Binding buffer per well. Add 100 μl of the mix into each well on a 96-well plate. Incubate overnight at 4 C at rpm to bind the proteins to the plate. As a positive control, in a separate well mix 5 μl of the provided phosphoprotein with 95 μl of Binding buffer. 2. Blocking the wells Remove solution from wells, add 150 μl of the Blocking buffer into each well and incubate 2-3 minutes. Remove the solution and add 150 μl of the Blocking buffer again and incubate for 30 min while shaking at rpm at room temperature. *At this stage, any additional manipulations can be carried out (e.g. kinase/phosphatase assay, inhibitor screening, etc.). Make sure to wash the wells 3x with the 1x TBST after each manipulation.* 3. pimago incubation In a clean tube, prepare a 1 to 100 mixture of the pimago reagent in the pimago buffer (1 μl of the reagent for every 100 μl of buffer). Empty the wells and add 100 μl per well of the prepared pimago mix. Incubate 1 hour at rpm at room temperature. 4. Washing the wells Empty the wells and add 150 μl of the pimago buffer into each well; incubate 2-3 minutes at rpm. Remove the buffer and repeat the washing step two more times with the pimago buffer for a total of 3 washes. Remove the solution and incubate the wells with 150 μl of the Blocking buffer for 15 min at rpm at room temperature. 5. Incubation with avidin-fluor In a clean tube, prepare 1 to 100 mixture of avidin-fluor in the Blocking buffer (1 μl of avidin-fluor in 100 μl of Blocking buffer). Empty the wells and add 100 μl per well of the prepared avidin-fluor in blocking solution. Incubate the plate in the dark for 1 hour at rpm at room temperature. 6. Washing the wells Empty the wells and add 150 μl of 1x TBST into each well; incubate 2-3 minutes at rpm and remove the solution. Repeat the washing step with TBST two more times for a total of three washes. Empty the wells. 7. Signal detection The signal can be detected using any fluorescence imager with an appropriate filter. The number listed with the provided avidin-fluor reagent represents the absorption maximum (emission wavelength) for that fluorophore. 5

6 Components Provided: Protocol for on 96-well Plate using Avidin-HRP Phosphorylated protein (β-casein) as a control (mix 5 μl with 95 μl Binding buffer; store at 4ºC); Blocking buffer (store at 4ºC); pimago buffer (store at 4 to 25ºC); Binding buffer (store at 4ºC); pimago reagent (store at 4ºC); avidin-hrp (store at 4ºC); Colorimetric Detection Substrates A and B (store at 4ºC); Stop Solution (store at 4 to 25ºC); 96-well plate for fluorescence detection. Need to prepare: 1x TBST (Tris-buffered saline with 0.1% Tween 20). PROTOCOL 1. Binding of samples to the 96-well plate Prepare a protein solution of your sample (phosphoprotein or substrate of interest) in Binding buffer. A dephosphorylated (e.g. alkaline phosphatase-treated or without ATP) form of the sample should also be prepared as a reference. If protein amount is known, use 10 to 500 ng of the protein or mixture of proteins per 100 μl of Binding buffer per well. Add 100 μl of the mix into each well on a 96-well plate. Incubate overnight at 4 C at rpm to bind the proteins to the plate. As a positive control, in a separate well mix 5 μl of the provided phosphoprotein with 95 μl of Binding buffer. 2. Blocking the wells Remove solution from wells, add 150 μl of the Blocking buffer into each well and incubate 2-3 minutes. Remove the solution and add 150 μl of the Blocking buffer again and incubate for 30 min while shaking at rpm at room temperature.. *At this stage, any additional manipulations can be carried out (e.g. kinase/phosphatase assay, inhibitor screening, etc.). Make sure to wash the wells 3x with the 1x TBST after each manipulation.* 3. pimago incubation In a clean tube, prepare a 1 to 100 mixture of the pimago reagent in the pimago buffer (1 μl of the reagent for every 100 μl of buffer). Empty the wells and add 100 μl per well of the prepared pimago mix. Incubate 1 hour at rpm at room temperature. 4. Washing the wells Empty the wells and add 150 μl of the pimago buffer into each well; incubate 2-3 minutes at rpm. Remove the buffer and repeat the washing step two more times with the pimago buffer for a total of 3 washes. Remove the solution and incubate the wells with 150 μl of the Blocking buffer for 15 min at rpm at room temperature. 5. Incubation with avidin-hrp In a clean tube, prepare 1 to 100 mixture of avidin-hrp in the Blocking buffer (1 μl of avidin-hrp in 100 μl of Blocking buffer). Empty the wells and add 100 μl per well of the prepared avidin-hrp in blocking solution. Incubate the plate in the dark for 1 hour at rpm at room temperature. 6. Washing the wells Empty the wells and add 150 μl of 1x TBST into each well; incubate 2-3 minutes at rpm and remove the solution. Repeat the washing step with TBST two more times for a total of three washes. Empty the wells. 7. Signal detection For normal and high concentrations of the proteins (majority of in vitro samples), use the provided colorimetry-based detection system. Prepare 9 to 1 mixture of the Colorimetric Substrates A and B (has to be made fresh each time before detection), and add 100 μl to each well. Shake the plate until satisfied with signal solution will turn green if signal is present (usually 1-2 min), then add 150 μl of the Stop solution to stop the HRP-substrate reaction. Read the plate at 415 nm in a plate reader. * Alternatively, any other peroxidase substrate can be used (chromogenic or chemiluminescent). However, for chemiluminescence-based detection, a different plate with non-transparent walls must be used. * 6

7 RELATED PRODUCTS Each box contains 10 cassettes GEL PERCENTAGE 2 WELL 12 WELL 17 WELL 8% NXG00812 NXG00827 Teo-Tricine SDS 10 x 10 cm cassette Compatible with SureLock TM tanks 10% NXG01012 NXG % NXG01212 NXG % NXG01612 NXG % (Gradient) NXG40812 NXG % (Gradient) NXG41212 NXG % (Gradient) NXG42002 NXG42012 NXG % BCG00812 BCG00827 Teo-Tricine SDS 8 x 10 cm cassette Compatible with BioRad tanks 10% BCG01012 BCG % BCG01212 BCG % BCG01612 BCG % (Gradient) BCG40812 BCG % (Gradient) BCG41212 BCG % (Gradient) BCG42012 BCG42027 Each box contains 10 cassettes GEL PERCENTAGE 12 WELL 17 WELL Bis-Tris 10 x 10 cm cassette Compatible with SureLock TM tanks 8% NBT00812 NBT % NBT01012 NBT % NBT01212 NBT % (Gradient) NBT41212 NBT41227 LUMIBLUE ECL SOLUTION CAT. # SENSITIVITY 1ºAB DILUTION RANGE (from 1mg/ml stock) 2º AB DILUTION RANGE (from 1mg/ml stock) COMPETITOR ECL SOLUTIONS Express ECLS0250 (250 ml) ECLS0500 (500 ml) low pg (~20 pg) 1:100-1:5,000 1:1,000-1:15,000 ECL Pierce (Thermo) Amersham ECL Start (GE) Pico ECLP0250 (250 ml) ECLP0500 (500 ml) low pg (~2pg) 1:500-1:5,000 1:20,000-1:100,000 Supersignal Pico (Thermo) Extra ECLA0250 (250 ml) ECLA0500 (500 ml) high fg (~500 fg) 1:1,000-1:15,000 1:25,000-1:150,000 Pierce ECL Plus (Thermo) Amersham ECL Plus/Prime (GE) Extended ECLD0100 (100 ml) ECLD0200 (200 ml) mid fg (~150fg) 1:5,000-1:50,000 1:50,000-1:250,000 Supersignal Dura (Thermo) ECL Prime (GE) Extreme ECLM0100 (100 ml) ECLM0200 (200 ml) low fg (~50 fg) 1:5,000-1:100,000 1:100,000-1:500,000 Supersignal Femto (Thermo) ECL Select (GE) NXB86500 DCX-700 EPS-300X RunBlue Tris-Glycine Transfer Buffer. 10x, 500ml Dual Cool Mini-Vertical System Power Supply, 1-300V, 1-500mA 7

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