Calculator
Considerations of Preparing Gel Electrophoresis Procedures
by K Chico, Jessica Piczon
Gel electrophoresis stains are chemicals used to visualize and identify fragmented proteins, DNA, and RNA in a gel. Stains for nucleic acids are typically fluorescent which can intercalate into their double helix structure. Common stains for proteins include Coomassie blue, silver, and fluorescent stains. Alternatively, negative staining can be used to stain the gel background white, leaving the transparent protein bands starkly visible. Total protein can be determined via colorimetric methods through Coomassie Blue, silver, and fluorescent staining.
Fluorescent methods are increasingly sensitive and can also be used to assess protein quantitation and posttranslational modifications. Staining with two or more different stains can be performed in series to provide an even greater understanding of the protein profile. The choice of stain and protocol depends on the necessary level of quantitation, available instrumentation, and downstream analytical procedures. Laboratory budget or curiosity may be the ultimate determinant of the chosen protocol.
Historically, gel staining protocols required several sequential steps. In essence, gels are first fixed to immobilize protein and remove sodium dodecyl sulfate (SDS), buffers, or other contaminants. Next, the gel is stained with dye or silver. Lastly, the gel quenched or destined to reduce background noise. Not only are there hundreds of variations of gel staining methods in the literature, nowadays many kits are available that offer exceedingly rapid protocols. Regardless, product manuals, material safety data sheets, and open-source literature may be needed to fully optimize a desired staining method. Common features of all successful gel staining protocols are established in good laboratory practices, including cleanliness, careful manipulations, and attention to detail. Some key considerations in staining are listed below.
Reagents: All water used should be pure, deionized, or double distilled.
Dishware: Literature suggests that staining should be done with a polycarbonate or polypropylene dish, however in many cases a glass dish will work. Dishware should be cleaned with 70-100% ethanol or methanol followed by a water rinse. Using a slightly larger dish will allow greater ease in handling.
Handling the Gel: In some protocols, the gel should float freely, neither stuck to the dishware nor floating on top of the fixing, washing, and staining solutions. Not only are gels fragile, but they are sticky. They can stick to the dish even in a buffer, which could pose a problem since a gel tear could ruin an experiment. For some staining protocols, a layer of saran wrap can first be placed in the dish to act as a liner. The stain can be poured on top of the saran wrap, and then the gel can be placed face down onto the stain. In this technique, the stain sticks to the gel by surface tension, which is typically an easier process than trying to deposit the stain directly onto the gel. Since saran wrap is transparent, there is no need to remove it prior to imaging, and instead of handling the gel, edges of the saran wrap can be handled instead.
Incubation: Some stains, like fluorescent stains, are light sensitive. At this point and beyond, stained gels should remain protected from light so that the dyes do not get exhausted prior to imaging. For this, aluminum foil is the most practical option. Gels should be incubated on a rocking platform or an orbital shaker at moderate speed. Many protocols have stated incubation times, though usually staining will be apparent within minutes. In some cases, increasing incubation times can provide greater sensitivity.
Ladder: If working with a low volume of protein, make sure to add a low volume of ladder so that the ladder does not overshadow the sample upon visualization.
Buffers: Most buffers, like tris-borate-EDTA (TBE) have a much higher level of buffering capacity than what is needed, so buffers can often be diluted two- to five-fold. Using a lower buffer concentration saves money and reduces heat generation. Sometimes an undiluted buffer must be used, especially when a really high resolution is required.
Gel visualization: If a stacking gel is used, it should be removed prior to visualization. If a commercial, pre-poured gel is used, the wells and the foot of the gel should be trimmed to reduce staining deposition artifacts, particularly if persisting SDS from the sample buffer remains in the gel. Though gels can be reused, be wary of residual stains that may compromise results, particularly with fluorescent stains.
Chapter 31 Protein Gel Staining Methods: An Introduction and Overview
Practical tips for staining nucleic acid PAGE gels without rips! & more tips on using SYBR gold, etc
Original created on August 28, 2023, last updated on August 28, 2023
Tagged under: electrophoresis, agarose, fluorescence, DNA, optimization, staining
Gel Staining
Comparison of Gelite™ Safe (1:25,000X dilution) and GelRed® (1:10,000X dilution) in precast gel staining using 1% agarose gel in TBE buffer. Two-fold serial dilutions of 1 kb DNA ladder were loaded in the amounts of 100 ng, 50 ng, 25 ng, 12 ng, 6 ng, 3 ng, 1.5 ng, and 0.7 ng from left to right. Gels were imaged using a 300 nm transilluminator in ChemiDoc™ Imaging System (Bio-Rad®).
Fluorescent methods are increasingly sensitive and can also be used to assess protein quantitation and posttranslational modifications. Staining with two or more different stains can be performed in series to provide an even greater understanding of the protein profile. The choice of stain and protocol depends on the necessary level of quantitation, available instrumentation, and downstream analytical procedures. Laboratory budget or curiosity may be the ultimate determinant of the chosen protocol.
Standard Steps and Considerations
Historically, gel staining protocols required several sequential steps. In essence, gels are first fixed to immobilize protein and remove sodium dodecyl sulfate (SDS), buffers, or other contaminants. Next, the gel is stained with dye or silver. Lastly, the gel quenched or destined to reduce background noise. Not only are there hundreds of variations of gel staining methods in the literature, nowadays many kits are available that offer exceedingly rapid protocols. Regardless, product manuals, material safety data sheets, and open-source literature may be needed to fully optimize a desired staining method. Common features of all successful gel staining protocols are established in good laboratory practices, including cleanliness, careful manipulations, and attention to detail. Some key considerations in staining are listed below.
Reagents: All water used should be pure, deionized, or double distilled.
Dishware: Literature suggests that staining should be done with a polycarbonate or polypropylene dish, however in many cases a glass dish will work. Dishware should be cleaned with 70-100% ethanol or methanol followed by a water rinse. Using a slightly larger dish will allow greater ease in handling.
Handling the Gel: In some protocols, the gel should float freely, neither stuck to the dishware nor floating on top of the fixing, washing, and staining solutions. Not only are gels fragile, but they are sticky. They can stick to the dish even in a buffer, which could pose a problem since a gel tear could ruin an experiment. For some staining protocols, a layer of saran wrap can first be placed in the dish to act as a liner. The stain can be poured on top of the saran wrap, and then the gel can be placed face down onto the stain. In this technique, the stain sticks to the gel by surface tension, which is typically an easier process than trying to deposit the stain directly onto the gel. Since saran wrap is transparent, there is no need to remove it prior to imaging, and instead of handling the gel, edges of the saran wrap can be handled instead.
Incubation: Some stains, like fluorescent stains, are light sensitive. At this point and beyond, stained gels should remain protected from light so that the dyes do not get exhausted prior to imaging. For this, aluminum foil is the most practical option. Gels should be incubated on a rocking platform or an orbital shaker at moderate speed. Many protocols have stated incubation times, though usually staining will be apparent within minutes. In some cases, increasing incubation times can provide greater sensitivity.
Ladder: If working with a low volume of protein, make sure to add a low volume of ladder so that the ladder does not overshadow the sample upon visualization.
Buffers: Most buffers, like tris-borate-EDTA (TBE) have a much higher level of buffering capacity than what is needed, so buffers can often be diluted two- to five-fold. Using a lower buffer concentration saves money and reduces heat generation. Sometimes an undiluted buffer must be used, especially when a really high resolution is required.
Gel visualization: If a stacking gel is used, it should be removed prior to visualization. If a commercial, pre-poured gel is used, the wells and the foot of the gel should be trimmed to reduce staining deposition artifacts, particularly if persisting SDS from the sample buffer remains in the gel. Though gels can be reused, be wary of residual stains that may compromise results, particularly with fluorescent stains.
| Buffer Preparation and Recipes: | Tools: |
Products
Table 1. Nucleic acid stains for agarose and polyacrylamide gel electrophoresis
| Product ▲ ▼ | Ex (nm)¹ ▲ ▼ | Filter² ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
| Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution* | 254 mn | Long path green filter | 1 mL | 17590 |
| Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution* | 254 mn | Long path green filter | 100 µL | 17604 |
| Helixyte™ Gold Nucleic Acid Gel Stain *10,000X DMSO Solution* | 254 mn | Long path green filter | 1 mL | 17595 |
| Gelite™ Green Nucleic Acid Gel Staining Kit | 254 nm or 300 nm | Long path green filter | 1 Kit | 17589 |
| Gelite™ Orange Nucleic Acid Gel Staining Kit | 254 nm or 300 nm | Long path green filter | 1 Kit | 17594 |
| Gelite™ Safe DNA Gel Stain *10,000X Water Solution* | 254 nm, 300 nm or 520 nm | Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters | 100 µL | 17700 |
| Gelite™ Safe DNA Gel Stain *10,000X Water Solution* | 254 nm, 300 nm or 520 nm | Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters | 500 µL | 17701 |
| Gelite™ Safe DNA Gel Stain *10,000X Water Solution* | 254 nm, 300 nm or 520 nm | Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters | 1 mL | 17702 |
| Gelite™ Safe DNA Gel Stain *10,000X Water Solution* | 254 nm, 300 nm or 520 nm | Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters | 10 mL | 17703 |
| Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution* | 254 nm, 300 nm or 520 nm | Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters | 100 µL | 17704 |
Table 2. ReadiUse™ DNA ladders
| Cat# ▲ ▼ | Product Name ▲ ▼ | Unit Size ▲ ▼ |
| 60050 | ReadiUse™ 1 Kb Plus DNA Ladder | 100 µL |
| 60051 | ReadiUse™ 1 Kb Plus DNA Ladder | 2x250 µL |
| 60055 | ReadiUse™ GeneRuler 1 kb DNA Ladder | 100 µL |
| 60056 | ReadiUse™ GeneRuler 1 kb DNA Ladder | 2x250 µL |
| 60070 | ReadiUse™ 100 bp DNA Ladder | 100 µL |
| 60071 | ReadiUse™ 100 bp DNA Ladder | 2x250 µL |
References
Chapter 31 Protein Gel Staining Methods: An Introduction and Overview
Practical tips for staining nucleic acid PAGE gels without rips! & more tips on using SYBR gold, etc
Original created on August 28, 2023, last updated on August 28, 2023
Tagged under: electrophoresis, agarose, fluorescence, DNA, optimization, staining