![]() 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) Sample Preparation: Cell Lysate and Conditioned Media Cells from 10 cm plates are are collected at 100 confluence, Day 0, in 1 mL of Western Lysis Buffer (WLB). 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. This page describes common challenges encountered when lysing cells and extracting proteins prior to Western blotting.When pipetting supernatant following centrifugation, take care not to disturb the pellet.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.Place Petri dish on ice and rinse twice with ice cold 1X PBS. Perform all lysate preparation steps at 4☌. Cells should be harvested at 80 confluency.We recommend adding 0.8 to 1 ml NP40 Cell Lysis. Protein concentration too low as seen on Coomassie-stained membrane Wash cells in the dish with ice-cold PBS and aspirate off PBS (1X). Non-Linear Standard Curve (for Colorimetric Assays for Total Protein).Variability in Measured Total Protein Concentration Between Replicates.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: 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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