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Cell lysates - Rinse cells with PBS, making sure to remove any remaining PBS before adding the lysis buffer. Solubilize cells at 4 x 107 cells/mL in 1x Lysis Buffer (we recommend adding protease and phosphatase inhibitors to lysis 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 100-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. Phospho-EphB3 ELISA 5 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 control then subtract the average blank optical density. i. Positive Control A431 cells were treated with recombinant human EGF at 37 °C for 10 min. Solubilize cells at 4 x 107 cells/mL in lysis 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 P-5 Phospho-EphB3 ELISA 10 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: Untreated A431 EGF treated A431 O D =4 50 n m 0 1 2 3 4 Phospho-EphB3 ELISA 11 X