Is Coomassie stain quantitative?
CBB may thus be considered as a very specific protein stain, eminently suited both to cytological observation and quantitative microdensitometry.
Is Coomassie Blue quantitative?
Two-dimensional electrophoresis in combination with Coomassie Blue staining was refined for use as a quantitative method. Microcomputer software was developed for use with the IBM AT and compatible computers for analyzing the gels.
What is the advantage of using Coomassie Blue over these other methods?
Coomassie dye staining is especially convenient because it involves a single ready-to-use reagent and does not permanently chemically modify the target proteins.
What is the role of SDS in an SDS-PAGE?
SDS is a detergent that is present in the SDS-PAGE sample buffer where, along with a bit of boiling, and a reducing agent (normally DTT or b e t a beta beta-ME to break down protein–protein disulfide bonds), it disrupts the tertiary structure of proteins. This brings the folded proteins down to linear molecules.
Why is Coomassie Blue used in SDS-PAGE?
Coomassie blue dyes are a family of dyes commonly used to stain proteins in SDS-PAGE gels. The gels are soaked in dye, and excess stain is then eluted with a solvent (“destaining”). This treatment allows the visualization of proteins as blue bands on a clear background.
Why is Coomassie blue used in SDS-PAGE?
Which staining mechanism is more preferable Coomassie Brilliant Blue or silver staining give your reasons?
All Answers (15) Coomassie is much faster but 10-100XX less sensitive than silver stain. If your band is faint you should use silver, but its also a bit more tedious.
What does SDS-PAGE determine?
SDS-PAGE is a reliable method for determining the molecular weight (MW) of an unknown protein, since the migration rate of a protein coated with SDS is inversely proportional to the logarithm of its MW.
What does Coomassie Blue staining tell us?
Coomassie brilliant blue stain offers high sensitivity, low background, large linear range, and ease of use for the identification of proteins separated by gel. It is the most commonly used staining technique for the quantification of proteins after gel electrophoresis.
Can Coomassie Brilliant Blue interfere in SDS-PAGE?
CBB can also bind SDS (that detergent we used earlier to solubilize and negatively-charge the proteins) which can mess up results. So you want to wash your gel in water before staining to remove the SDS.
Is silver staining more sensitive than Coomassie?
Coomassie Blue staining is approximately 50-fold less sensitive than silver staining, however due to its simplicity binding Coomassie Blue is preferred. A final consideration in the choice of staining agent is the ability to remove the stain (destain).
Is SDS-PAGE qualitative or quantitative?
Immunoblot SDS-PAGE analysis is a valid method of obtaining a qualitative and quantitative analysis of urinary cyst-C. This method presents unique information about a previously unknown 70 KDa cyst-C form. The assay may offer potential diagnostic information not available with immunometric method.
What are the limitations of SDS-PAGE?
An obvious limitation of SDS-PAGE resides in its deliberate denaturation of proteins prior to electrophoresis. Enzymatic activity, protein binding interactions, detection of protein cofactors, etc. generally cannot be determined on proteins isolated by SDS-PAGE.
How can we improve protein separation in SDS-PAGE?
Try increasing the percentage of SDS in both the gel and the migration buffer (x2 or x3), you reduce the migration time and the protein bands will be well separated! Add a prestained protein ladder next to your sample on a 4-20% SDS-PAGE.
What is purpose of Coomassie Blue in SDS-PAGE?
Popular Answers (1) Coomassie Blue stain is used to stain the protein bands in polyacrylamide gels. One common way to use it is to dissolve the dye in a mixture of methanol, acetic acid, and water. This stain will permeate the gel, stain the protein, and also fix the protein in place.
Why do we use Coomassie blue in SDS-PAGE?
What is the difference between Coomassie blue and silver staining?
Coomassie blue staining is a quick, simple, and affordable method for detecting proteins on gels. It has a detection limit of ~ 0.1–0.5 μg protein, sensitive enough for most daily needs. Silver staining has greater sensitivity, but involves many more steps and solutions (see Silver Staining of SDS-polyacrylamide Gel ).
How much protein can be detected in a Coomassie stain?
3-10 microgram of protein can be detected usually within 5 minutes in Coomassie stains. With additional water-based de-staining, as little as 7 ng of protein (BSA) can be detected.
What is the difference between pageblue and Coomassie blue dyes?
Coomassie blue dyes bind proteins quantitatively within a certain protein range allowing for densitometry analysis. PageBlue protein stain can deliver a dynamic range of ~5ng to ~500ng.
Does the concentration of Coomassie matter for dye concentration?
The concentration of the Coomassie shouldn’t matter too much. Only so much of the dye in your solution can be bound by the proteins (which you don’t have a lot of) anyway.