EGFR ELISA 试剂盒

• Simultaneously measure Phosphorylated protein and pan protein in one experiment (for normalization purpose)
• Screen numerous different cell lysates without performing a Western Blot analysis
• Minimal hands-on time, convenient, and non-radioactive material

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Anti-Phospho Antibody
• Anti-Pan Antibody
• HRP-Conjugated Secondary Antibody
• Streptavidin-Conjugated HRP
• Assay Diluent
• TMB One-Step Substrate
• Stop Solution
• Lysis Buffer
• Positive Control Sample

• Distilled or deionized water
• 100 mL and 1 liter graduated cylinders
• Tubes to prepare sample dilutions
• Protease and Phosphatase inhibitors
• Precision pipettes to deliver 2 μL to 1 mL volumes
• Adjustable 1-25 mL pipettes for reagent preparation
• Benchtop rocker or shaker
• Microplate reader capable of measuring absorbance at 450 nm

1. Prepare all reagents and samples as instructed in the manual.
2. Add 100 μL of sample or positive control to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared primary antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared 1X HRP-Streptavidin to each well.
7. Incubate 1 h at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Item E, Assay Diluent should be diluted 5-fold with deionized or distilled water before use. Human Phospho-EGFR (Tyr 1045) and pan EGFR ELISA Kit Protocol 6
   3. Preparation of Positive Control: Briefly spin the Positive Control vial of Item K. Add 400 µL 1x Assay Diluent (Item E, Assay Diluent should be diluted 5-fold with deionized or distilled water before use) into Item K vial to prepare Positive Control Solution (P-1) (See i. Positive control of part IX.for a typical result). Dissolve the powder thoroughly by a gentle mix. Pipette 320 µL 1x Assay Diluent into each tube. Add 80 µL prepared P-1 Solution from the vial of Item K, into a tube with 320 µL 1x Assay Diluent to produce a dilution series (shown below). Mix each tube thoroughly before the next transfer. 1x Assay Diluent serves as the background.
   4. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 mL of Wash Buffer Concentrate into deionized or distilled water to yield 400 mL of 1x Wash Buffer.
   5. Briefly spin the anti-phospho-EGFR (Tyr 1045) (Item C) before use. Add 100 µL of 1x Assay Diluent into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days. It can be used within one month if store at -80 °C. Avoid repeated freeze-thaw cycles). The detection antibody P (1) P (2) P (3) P (4) P (5) 0 80 µL 400 µL 1x Assay Diluent 80µl 80 µL 80 µL Human Phospho-EGFR (Tyr 1045) and pan EGFR ELISA Kit Protocol 7 concentrate should further be diluted 60-folds with 1x Assay Diluent and used in step 4 of Part VII Assay Procedure.
   6. Briefly spin the HRP-conjugated anti-rabbit IgG (Item D-1), before use. Pipette up and down to mix gently. HRP- conjugated anti-rabbit IgG concentrate should be diluted 1,000-folds with 1x Assay Diuent.
   7. Briefly spin the Detection Antibody vial (Item L) before use. Add 100 µL of 1x Assay Diluent into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days. It can be used within one month If store at -80 °C. Avoid repeated freeze-thaw cycles). The detection antibody concentrate should be diluted 200-folds with 1x Assay Diluent and used in step 4 of Part VI Assay Procedure.
   8. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use since precipitation may form during storage. HRP-Streptavidin concentrate should be diluted 600-fold with 1x Assay Diluent. For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 20 µL of HRP-Streptavidin concentrate into a tube with 12 mL 1x Assay Diluent B to prepare a 600-fold diluted HRP-Streptavidin solution (don’t store the diluted solution for next day use). Mix well.
   9. Cell Lysate Buffer should be diluted 2-folds with deionized Human Phospho-EGFR (Tyr 1045) and pan EGFR ELISA Kit Protocol 8 or distilled water before use (recommend to add protease and phosphatase inhibitors). VII.

Cell lysates - Rinse cells with PBS, making sure to remove any remaining PBS before adding the Cell Lysate Buffer. Solubilize cells at 4 x 107 cells/mL in 1x Cell Lysate Buffer (we recommend adding protease and phosphatase inhibitors to Cell Lysate Buffer prior to sample preparation). Pipette up and down to resuspend and incubate the lysates with shaking at 2 - 8° C for 30 minutes. Human Phospho-EGFR (Tyr 1045) and pan EGFR ELISA Kit Protocol 5 Microcentrifuge at 13,000 rpm for 10 minutes at 2 - 8° C, and transfer the supernates into a clean test tube. Lysates should be used immediately or aliquoted and stored at -70 °C. Avoid repeated freeze-thaw cycles. Thawed lysates should be kept on ice prior to use.
For the initial experiment, we recommend to do a serial dilution testing such as 5-fold and 50-fold dilution for your cell lysates with Assay Diluent (Item E) before use.
Note: The fold dilution of sample used depends on the abundance of phosphorylated proteins and should be determined empiricallys. More of the sample can be used if signals are too weak. If signals are too strong, the sample can be diluted further.
Cell Lysate Buffer should be diluted 2-fold with deionized or distilled water before use (recommend to add protease and phosphatase inhibitors).

1. Bring all reagents to room temperature (18 - 25 °C) before use. It is recommended that all samples or Positive Control should be run at least in duplicate.
   2. Add 100 µL of each sample or positive control into appropriate wells. Cover well with plate holder and incubate for 2.5 hours at room temperature or over night at 4 °C with shaking.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 µL) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of 1x anti-phospho-EGFR (Tyr 1045) (see Reagent Preparation step 5) to corresponding well for detecting phospho-EGFR or 100 µL of 1x Biotinylated anti-EGFR (see Reagent Preparation step 7) to corresponding well (help normalize the results of phospho-EGFR from different cell lysate being compared) for detecting a pan EGFR. Incubate for 1.5 hour at room temperature with shaking. Human Phospho-EGFR (Tyr 1045) and pan EGFR ELISA Kit Protocol 9
   5. Discard the solution. Repeat the wash as in step3.
   6. Add 100 µL of 1,000-fold diluted HRP-conjugated anti-rabbit IgG (see Reagent Preparation step 6) to detect Rabbit phospho- EGFR (Tyr 1045) (corresponding well of adding Rabbit phospho-EGFR). Incubate for over night at 4 °C. Add 100 µL of 600 diluted HRP-Streptavidin (see Reagent Preparation step 8) to detect Biotinylatded EGFR antibody (corresponding well of adding Biotinylatded anti-EGFR antibody). Incubate for 1 hour at room temperature with shaking.
   7. Discard the solution. Repeat the wash as in step3.
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

ELISA data analysis: Average the duplicate readings for each sample or positive control then subtract the average blank optical density.
i. Positive Control A431 cells were treated with recombinant human EGF at 37 °C for 20 min. Solubilize cells at 2 x 107 cells/mL in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI Reagent Preparation for detail. Human Phospho-EGFR (Tyr 1045) and pan EGFR ELISA Kit Protocol 11 Assay Diluent Positive control dilution fold O D = 4 5 0 n m 0.01 0.1 1 10 2 10 50 250 1250
ii. Recombinant Human EGF Stimulation of A431 Cell Lines A431 cells were treated or untreated with 100 ng/mL recombinant human EGF for 10 min. Cell lysates were analyzed using this phosphoELISA and Western Blot. ELISA Phospho-EGFR (Tyr1045) EGFR O D =4 50 n m 0.0 0.5 1.0 1.5 2.0 2.5 3 3.5 4.0 Untreated A431 EGF treated A431 Human Phospho-EGFR (Tyr 1045) and pan EGFR ELISA Kit Protocol 12 Western-Blot hEGF 0 10 0 10 (Min) Anti-phospho-EGFR Anti-EGFR (Tyr1045) X