Choose an assay with a broader linear dynamic range.Increase or decrease lysate concentration until it is within the linear dynamic range of the selected assay.Protein concentration outside of linear dynamic range of assay Learn about infrared-based protein quantitation of cell lysates.Use infrared-based protein quantitation, which is compatible with a wider range of buffers than colorimetric assays.Verify that buffers used are compatible with chosen protein quantitation assay.Learn about assay-free infrared-based total protein quantitation using the Direct Detect® system.Switch to infrared-based protein quantitation, which is not time-dependent.Time before reading absorbance too long (for colorimetric assays) Learn about infrared-based protein quantitation.Switch to infrared-based protein quantitation, which directly measures amide bonds in protein chains.Incorrect protein standard selected (for colorimetric assays) Ensure accurate pipetting use properly calibrated pipettes.Scepter™ cell counter ordering informationīack to Top Variability in Measured Total Protein Concentration Between Replicates Possible Cause.Get tips on accurate cell counting using the Scepter™ handheld counter.If using dissociated/cultured cells, make sure cell counts are accurate.Store on ice for immediate use, or at -20C or -80C until needed. Transfer supernatant to a fresh tube and discard cell pellet. Clarify the lysate with a high speed spin in a microfuge at 4C, for 10 minutes at 12,000 rPM. When pipetting supernatant following centrifugation, take care not to disturb the pellet. Add tissue and 1-2 ml ice cold lysis buffer to dounce homogenizer, or sonicate in small tube.Increase time/power setting of sonication.Create lysate aliquots to minimize number of freeze-thaw cycles.Try using different inhibitor cocktails.Add or increase concentration of protease and phosphatase inhibitors.If sonicating, minimize total sonication time or insert breaks in between sonication pulses to minimize heat generation.Perform all lysate preparation steps at 4☌.Protein concentration too low as seen on Coomassie-stained membrane Non-Linear Standard Curve (for Colorimetric Assays for Total Protein).Variability in Measured Total Protein Concentration Between Replicates.Chop the tissue into small pieces (0.1g to 1g each) after. Variables affecting each of these steps are outlined below, as each could affect the sensitivity and reproducibility of the Western blot.Ĭlick on the symptoms to read about the possible causes and remedies: Protein Extraction from Tissue Place surgically resected tissue in pre-cooled (4C) normal saline. Total protein concentration must be determined for these cell lysates. Often, the first step of analyzing protein expression or protein-protein interactions is to obtain a cell or tissue sample, lyse the cells, and extract proteins using extraction reagents. Related Resources: Brochures | Application Notesīefore you start analyzing a protein sample by Western blotting, read our web page, “ Protein Preparation,” for technical and product information on this important part of the Western blotting workflow.
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