Protein Technology Topics: Summary of Protein Standards (Marker) Knowledge

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Protein Marker can be divided into: first, unpre-marked Marker, broad molecular protein standard, high molecular weight protein standard and low molecular weight protein standard; second, pre-dyed Marker is monochrome pre-dyed and multi-color pre-dyed.

In the western blot process, the molecular weight Marker is like a screw, although it is a small detail, but such a small detail has a negligible effect on the experimental results. The role of a member of the Western Blot reference family is mainly used to indicate the molecular weight of the protein band. Only the standard amount is accurate, and the experimental results are convincing. In addition, the protein standard also indicates successful transfer. Or the degree of electrophoresis of the protein on the gel, etc., so choosing the correct protein Marker is also one of the necessary conditions for the success of the western blot experiment.

In general, the molecular weight standard can be divided into two levels: undyed protein molecular weight standard and pre-stained protein molecular weight standard. The following is a small narrative about protein molecular weight standards:

I. Pre-mixed protein molecular weight standard

The unstained protein molecular weight standard is the simplest and most accurate one. Since there are no dye molecules or labeling molecules attached, the size shown is exactly the size of the protein, which is necessary to accurately determine the size of the protein. Most of the current Marker uses a premixed Marker to facilitate comparison of proteins of different sizes. Premixed Markers usually have several bands with doubled concentration as an indication, because the more bands you mix, the harder it is to remember, who knows which one is that! It counts up to the eye. So when you see the few thick bands, you can remember where it is. But remember, the small band is usually not so easy to see. In terms of choice, of course, it is best to choose at least one of the strips and the size of the target protein of your own, the closer the better. If your protein is unfortunately between two larger Marker bands, choose Marker. The premixed Marker is not as good as the pre-stained Marker (pre-stained). Because it is completely invisible during the electrophoresis process, it is necessary to wait until the final staining with the target protein to "open" and it is impossible to predict the experiment. It is entirely in the "post-knowing" style, of course, it is better than "unconsciously".

1 broad molecular weight protein standard

If you consider it from the laboratory as a whole, choose the range as wide as possible, and the distribution of the bands is relatively uniform, so that your protein can be easily judged in any interval, avoiding the danger of falling in a blank area. However, the more strips, the more you load each time. After getting Marker, if you buy a big package, you should consider packaging. Repeatedly freeze and thaw the risk of protein, needless to say. Also remember that the broad-spectrum Marker doesn't necessarily have to be seen in every single item, especially the Mini Cell. If the side is a major protein, then the small may have ran away, not the quality is not good.

2 high molecular weight protein standard

High molecular weight protein standards are often used in the detection of large molecular weight proteins.

3 low molecular weight protein standards

In general protein electrophoresis, the lower molecular standard molecular weight is more commonly used, and in addition to the effect of indicating the size of the protein, it can sometimes be used as a rough quantitative. The laboratory is used in low molecular weight protein standards for general protein electrophoresis.

Also included in the low molecular weight protein standard are particularly small protein standards, such as the separation of proteins or polypeptides below 30 kD. Among them, GE's protein standard of 6 bands from 2kD to 16kD is quite good. Another reminder is that if the color of the Marker strip is particularly small, it is necessary to load enough.

2. Pre-stained protein molecular weight standard

The pre-stained protein molecular weight is a mixture of some purified proteins that can be directly observed by electrophoresis or during membrane transfer by covalent coupling with the dye. The appearance of the molecular weight of the pre-stained protein facilitates our experiments. This protein molecular weight standard can help us monitor the electrophoresis and estimate the mobility during electrophoresis, after electrophoresis, and after transfusion. For example, the best resolution region for vertical electrophoresis is known. About 2/3 of the glue, if the pre-stained Marker is used, it can predict that the target protein will enter the optimal resolution zone and stop electrophoresis for the best resolution; if Marker electrophoresis is observed, the electrophoresis can be terminated in time; in addition, in Western Blot After transfer, the protein transfer membrane can be directly observed, and the molecular weight of the protein can be labeled on the membrane, so many laboratory personnel are attracted to purchase the pre-stained protein standard. It is worth noting that the pre-stained protein Marker is covalently coupled with the dye, so there may be some changes in the migration characteristics when electrophoresis under different buffer conditions, which may lead to some deviations, so it is not suitable for precise localization of proteins - but in most cases The Marker strip may not be exactly the same as our target protein. What we get is just a reference size relative to Marker's indication. Finally, we need Western to characterize, so if we don't want to distinguish strips of similar size, pre-dye Marker is still very useful and can also be used with unstained protein standards.

Pre-stained protein standards can be divided into two types: monochrome pre-dyed and multi-color pre-dyed. The difference is to help identify the size of a strip during the experiment. The more the Marker strip, the more reference value it has. The more difficult it is to distinguish between distinctions, it is better to distinguish them if they are distinguished by different colors. If you run out of the colorful Rainbow Marker in a dull electrophoresis experiment, you will be happy at this time! Monochrome Marker usually uses some of the bands to double the concentration to indicate its size. It is also important to note that since the transfer film is a condensed Marker on a white film, the amount of sample loading is relatively small, if you want to see a beautiful "rainbow" during electrophoresis, or Monochrome Marker often needs to add more Marker than the manual.

Third, Western-specific protein molecular weight standards

1 biotin labeling protein standard

When the unstained Marker is used in Western, it is necessary to use the coloring method such as Ponceau or SYPRO fluorescent protein staining solution to interfere with Western Blot before the imprinting, and then mark on the gel to finally "spelt" the final result. In the case of a pre-dyed Marker, the film is marked after the film is transferred, or the film is cut, and finally "spelled". If you want to display the results of the Marker directly on the final result, without working with a drawing or stitching, you should use the Western-specific Marker. Such as biotin-labeled protein standards. This method is mainly used in conjunction with the chemiluminescence method: biotin is labeled on the molecular weight of the protein, and the antibody with the antibiotic or the antibody coupled with the streptavidin can be simultaneously observed when the target protein band is detected by chemiluminescence. The corresponding molecular weight standards are illuminated together. Different from the convenient molecular weight of common protein molecular weight standards, the advantage of this method is that it has good correspondence with Marker and the target protein, and it is displayed on the same sheet without splicing, which is convenient for analysis. The disadvantage is that if the sample has a biotin background, the anti-biotin antibody may affect the results, and the reaction system is too complicated to interfere with the experimental results.

2 special labeling protein standard

In the modified protein standard, in addition to biotin labeling, protein standards with "tails" have emerged in recent years due to the enrichment and complication of downstream protein manipulation. For example, His-Tag, if each Marker band has a His-tag, it is easy to display the Marker band on the Western result by adding a His-Taq antibody to the secondary antibody. Very convenient, the problem is that there may be potential interference, such as the structure of the sample just like Histag. However, this can be detected by comparison.

Most of the protein standards commonly used in laboratories are from NEB, MBI, GE, Bio-Rad, Invitrogen, Novagen, Promega, MRESCO, Pierce Bio., etc. Bio-help 2-D protein MW Markers product recommendation: Marker for 2D Electrophoresis, ColorBurst(TM) Electrophoresis Marker, Myoglobin, Carbamylated 2D Electrophoresis Marker from equine heart