![]() In all of them, key factors for success, data analysis and interpretation are covered as well as links to additional educational resources. LI-COR has several other protocols to help you meet publication guidelines and requirements. Refer to Quantitative Western Blot Analysis with Replicate Samples to guide you in your experimental set up. Biological and technical replications should both be done, since they are both important but meet different needs. Replication is an important part of quantitative Western blot analysis and is used to confirm the validity of observed changes in protein levels. There is also detailed information on normalization calculations, analysis of replicates, and data interpretation. It includes step-by-step instructions on how to use Revert 700 stain. The Revert 700 Total Protein Stain Normalization protocol describes how to use Revert 700 Total Protein Stain for Western blot normalization and quantitative analysis. Revert 700 staining is imaged at 700 nm, and fluorescent signals are proportional to sample loading. Revert 700 Total Protein Stain is a near-infrared fluorescent membrane stain used for total protein detection and normalization. The figure on the right shows that Revert 700 Total Protein Stain provides highly efficient protein staining on nitrocellulose or Immobilon ®-FL PVDF membranes in under 10 minutes. This antibody-independent method corrects for variation in both sample protein loading and transfer efficiency, and monitors protein transfer across the blot at all molecular weights. Because this internal loading control uses the combined signal from many different sample proteins in each lane, error and variability are minimized. Revert™ 700 Total Protein Stain provides linear, proportional signal across a broad range of sample concentrations.Īfter transfer, but prior to immunodetection, the membrane is treated with a total protein stain to assess actual sample loading across the blot. You should make sure that the signal intensity of the total protein stain is moderate, without saturation or low signal-to-noise ratios. The relative fluorescence intensity is the average of triplicate values. C32 cell lysate was separated in 4-12% Bis-Tris gels and transferred to nitrocellulose membrane. Revert 700 remained linear from 1 to 60 μg of cell lysate. Above 20 μg of cell lysate, housekeeping proteins began to saturate and did not exhibit a linear signal response. A linear response is required for quantitation. Revert 700 Total Protein Stain has a wider linear range than housekeeping proteins. And, it must be compatible with downstream immunodetection of your blot. It should also correct for variation at all points in the Western blot process, including gel loading and transfer to membrane. A total protein stain should produce a linear increase in signal intensity in response to increasing protein concentration. ![]() Unlike housekeeping proteins, total protein staining does not require validation for each experimental context. This more direct approach may increase the accuracy of normalization. For each lane, the sum of all the signal intensities of all the proteins in the lane is used for normalization. ![]() Total protein staining is a direct measure of the total amount of sample protein in each lane. ![]() While you have choices for your Western blot normalization strategy – you can still use housekeeping proteins as long you have validated that their expression is not changing – total protein staining detection is becoming the "gold standard" for normalization of protein loading. ![]() In the instructions to authors for the Journal of Biological Chemistry, they state: ![]()
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