Staining methods for 1D gels and blots and their evaluation in LabImage 1D

Different staining methods after 1D gel electrophoresisin laboratory diagnostics, there are different methods for quantifying protein or DNA/RNA bands after gel electrophoretic separation.The first step is to make the bands that are still invisible visible, so that in a second step their intensity can be displayed as a numerical value using a corresponding calibration curve.This second step is carried out by evaluating the corresponding image file using the LabImage 1D software created by Kapelan Bio-Imaging.The accuracy and meaningfulness of the data obtained is directly related to the selected staining method and the quality of staining implementation.The more specific the coloring is and the cleaner the work, the more accurate are the results derived from it.For more general staining of proteins, DNA, or RNA directly on the gel, the following methods are most commonly used.Here, the bands separated according to size are stained without a specific target molecule being exactly detected.These methods primarily help to get a quick overview within a large sample volume.An exact quantification with quantity determination is rather difficult due to the non-specific staining as well as a wide scatter and background staining, but often not necessary.It is often more about an initial sorting into positive and negative samples.Such fast scans can be carried out quickly and easily using automated program loops with LabImage 1D, even with very large sample volumes.

1. Coomassie Blue

Coomassie blue staining is a simple and inexpensive staining method for visualizing proteins. The dye binds to basic and aromatic amino acid residues, such as those found in e.g. arginine, histidine, lysine or tryptophan. Coomassie blue staining is a final step, so no further analysis can be performed after the sample has been stained. Due to the low price and the easy handling, this staining is ideal when there is a large throughput of samples. Because the tracks often do not run straight, the “curved track” track mode makes sense. The band detection delivers very good results with the standard modes. The rolling ball is mainly used for background correction. The background level is rather low, so the correction is not very deep. But you should pay attention to the band limits. These have a very clear influence on the band volume. Since LabImage determines the structure of the gels/blots itself, no inversion is required. This would be recognizable by the band peaks running downwards in the profile. Here you can carry out the inversion in the preprocessing area.

2. Silver Staining

Silver staining is mostly used to detect proteins to which the applied silver ions attach with high affinity. However, they also bind to phosphate groups that are part of RNA and DNA, so silver can in principle also be used to stain DNA or RNA samples. However, the sensitivity here is so low that other staining methods are preferable. For proteins, the silver stain is up to 10 times more sensitive than the Coomassie blue stain and can therefore also detect very small amounts of protein better. However, the implementation is more complex and expensive. Silver staining often leads to tapering, non-straight tracks. The best way to do this is to use the “curved track” track mode. Since the tracks often get wider towards the bottom, the total width must be set so that the width at the front of the track dictates the total width. Overlapping tracks are not allowed in LabImage by default. Background correction should use Rolling Ball mode. Depending on the structure, “minimum to minimum” also makes sense. Here the rise to the next minimum can be adjusted via the slope parameter.

3. Ethidium bromide staining

Ethidium bromide staining is a specific method for detecting nucleic acids after gel electrophoresis. The dye attaches itself to the DNA or RNA strands and emits fluorescent light when the gel is exposed to UV light. The method is very sensitive and can already detect very small amounts of DNA or RNA. At the same time, it is relatively quick and easy to carry out. However, ethidium bromide is a carcinogenic agent, so appropriate caution and special protective measures are warranted. In addition, ethidium bromide can damage the sample if applied imprecisely, thereby affecting the band intensity. Despite its carcinogenic effect, the ethidium bromide staining of DNA gels is a very widespread method and can be processed very robustly in the LabImage 1D. As with all other methods, it is important for the evaluation that the evaluation ROI (region of interest) is set very briefly. Traces and bands are found very easily automatically, so that little or no manual reworking is required. With the background correction, you can even work with a baseline due to the rather homogeneous background. The “rolling ball” also delivers good results here.

4. SYBR Green stain

SYBR Green binds specifically to nucleic acids and is used in particular for the detection of DNA. With a few swabs, however, RNA can also be easily detected. The dye lodges in the strands of the molecules and emits a fluorescent light when exposed to UV light. The method is very sensitive and can detect very small amounts of DNA. Compared to ethidium bromide staining, SYBR Green is less toxic but more expensive. Because SYBR Green detection is very sensitive, this method is also prone to contamination, which can result in high background noise. The “rolling ball” should therefore also be used as a method for background correction. Traces and bands can be detected largely automatically. Since the gangs can often be at an angle, you can use the grimace tool to separate gangs. This virtually corrects the position of the bands in such a way that no bands “run into each other” in the profile and can therefore be separated cleanly. This is particularly important when quantifying bands. The methods explained are mostly used for a first pre-selection. However, when it comes to detecting specific proteins, DNA or RNA molecules in a sample, detection systems must be used that are specific and allow quantification of well-defined parts of the total sample. The result of such methods allows a quantity determination of the target molecule contained within a sample using a calibration curve. With the help of LabImage 1D, the analysis of corresponding image files can also be automated according to constant criteria, so that large amounts of data can be processed quickly and reproducibly.

There are basically two different options for specific detection methods:

  1. Specific detection directly on the gelImmunostaining can be performed to detect specific proteins directly in the gel.The gel is incubated in a solution with specific antibodies that bind to the desired protein.In the second step, these antibodies are then labeled by a secondary antibody that carries a dye (usually fluorescent) or an enzyme, which ultimately makes the binding visible.Analogous to this, certain DNA or RNA strands are detected by hybridization.In this case, specific probes are used, which are more or less the counterpart of the molecule segment to be detected and also carry a dye or an enzyme in order to make the binding visible in the next step.
  2. Blotting procedure During blotting, the separated samples are transferred from the gel onto a nitrocellulose or PVDF membrane. Here, too, the specific molecule is detected by the respective special antibodies or probes. Blotting methods are usually more sensitive than direct detection methods in the gel, since they have a higher detection sensitivity. This is because transferring the proteins or nucleic acids from the gel to the blotting membrane allows for a higher concentration and purity of the target molecule. However, blotting methods are also more complex and therefore more expensive than direct detection methods on the gel. Western blotting is used to detect and characterize proteins, Southern blotting to detect specific DNA sequences, and Northern blotting to detect RNA sequences.

In summary, there are many methods to stain a gel after the sample has been separated. The choice of this method depends on the specific requirements and the availability of resources. At the end there is always an image with several or individual bands that can be quantified by measuring the optical density. A defined target value can be determined both qualitatively and quantitatively. LabImage 1D can carry out the corresponding evaluation automatically, so that a large sample throughput and constant comparison conditions are given. The accuracy of this quantification stands and falls with the sensitivity and specificity of the detection method used.