Western Blots
During the step where separated proteins from the gel are transfer an absorbent membrane, a methanol-based transfer buffer is commonly used. Methanol causes retinaland optic nerve damage, metabolic acidosis and dermatitis.
Ethanol may be an excellent substitute. McGill University replaced the methanol with ethanol and found that there is no difference in the detection of a phosphorylated protein when ethanol is used to make the transfer buffer.
Gel Electrophoresis
Widely used in DNA, RNA, protein
An electric field is applied across the gel, causing the negatively charged DNA/RNA/proteins to migrate across the gel towards the positive electrode. Smaller molecules migrate through the pores of the gel more readily vs larger molecules, hence you get separation by size.
Ethidium Bromide (EtBr) is used in gel electrophoresis. It is toxic and mutagenic. Under UV light, it fluoresces orange. UV light used to visualize results is harmful to your eyes. It’s also very economical.
Alternatives:
- GelRed - less mutagenic that EtBr and more shelf stable than SYBRsafe (below). Still uses UV light at for visualization (excitation @ 300nm and emission @ 595nm).
Sources discussing use, cost, sensitivity and comparisons: Huang et al., Haines et al., Haines et al., Cunha et al.. (For more information, contact @SvenLMB who uses Gel Red in his lab at Universitetet i Bergen) - GelGreen - Less mutagenic, but high acute toxicity. It is as sensitive as EtBr. It does not require UV light. Expensive.
Sources: Cunha et al., Haines et al., Haines et al., Evenson et al. - SYBRsafe - less mutagenic
Sources: Evenson et al. - Thiazole Orange - Cheap and sensitive. Visualized with blue light, but can cause skin and eye irritation.
Sources: Evenson et al., O’Neil et al. - Fastblast - not carcinogenic and doesn’t use UV light. Require several rinsing steps with tap water (more time consuming)
Sources: Fastblast SDS
Sensitivity
Toxicity
Ethidium Bromide is considered a mutagen, but some argue that its metabolites are the real cause of mutagenicity. Studies have shown that when metabolic enzymes are introduced, Salmonella TA98 and TA1537 experience significantly more mutations in their colonies. Other alternatives, such as GelRED and SYBR Safe, have also been found to be slightly mutagenic when metabolized.
Excitation
Ethidium Bromide emits an orange glow when exposed to certain wavelengths within the ultraviolet spectrum. This glow is caused by the absorption of light at 210 and 295 nm. Alternative stains such as GelRed and SYBR Safe also emit similar orange-red glows, but have added benefits. For example, GelRed can be visualized using ultraviolet transilluminators without the need to change filters, like Ethidium Bromide. SYBR Safe, on the other hand, can also be excited under blue light, which reduces the risks of DNA damage caused by UV light exposure. This makes SYBR Safe a safer option.
Immunohistochemistry
Triton x-100 is toxic to aquatic life and should be disposed as a hazardous waste to prevent Environmental damage. Replace with:
Tween-20 = Sigma SDS for that compound says it shouldn’t be released down the drain but reports no environmental effect. Triton surfactants can solubilize the lipid bilayers of bacterial cell membranes while Tween surfactants cannot.
Sorbitan derivatives (including poysorbates) - included in the EPA Safer Choice ingredients list as being of low environmental concern.
Also, Brij 35 (poly(oxyethylene) 23 lauryl ether), NP-40 or CHAPS (May also be toxic, environmental impact not checked)
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