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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. 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 1x Assay Diluent (Item E) before use. Phospho-Met (Tyr1234/1235) ELISA Kit Protocol 5 Note: The fold dilution of sample used depends on the abundance of phosphorylated proteins and should be determined empirically. 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).
ELISA data analysis: Average the duplicate readings for each sample or positive. i. Positive Control H1993 cells were cultured at 37 °C for 4 days. Solubilize cells at 4 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. Assay Diluent Positive control dilution series O D = 4 5 0 n m 0.01 0.1 1 10 P-1 P-2 P-3 P-4 0 Phospho-Met (Tyr1234/1235) ELISA Kit Protocol 10 ii. Recombinant Human HGF Stimulation of A431 Cell Lines A431 cells were treated or untreated with 50 ng/mL recombinant human HGF for 5 min. Cell lysates were analyzed using this phosphoELISA. Phospho-Met (Tyr 1234/1235) Pan Met O D =4 50 n m 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Untreated A431 HGF treated A431 iii. SENSITIVITY H1993 cells were cultured at 37 °C for 4 days. Solubilize cells at 4 x 107 cells/mL in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA and by Western blot. Immunoblots were incubated with anti-phospho-Met (Tyr1234/1235). Phospho-Met (Tyr1234/1235) ELISA Kit Protocol 11 A) ELISA 40 8 1.6 0.32 0.08 0 ( µg ) O D =4 50 n m 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 B). Western-Blot Analysis 50 25 12.5 6.25 3.13 1.56 0.78 0.39 0 (µg) Phospho-Met (Tyr1234/1235) ELISA Kit Protocol 12 X