How much Mercaptoethanol is in a sample buffer?
Add 50 µl of β-mercaptoethanol per 950 µl of sample buffer for a final concentration of 5% β-mercaptoethanol, 710 mM. As an alternative, dithiothreitol (DTT or Cleland's reagent) may be used at a final concentration of 350 mM (54 mg/ml). Dilute 1 part sample with 1 part Laemmli sample buffer.
When preparing SDS-PAGE sample buffer, you can use either beta-mercaptoethanol (BME) or dithiothreitol (DTT). For BME, use a concentration of 5% (about 100 mM). For DTT, use 5-10 mM.
2-Mercaptoethanol (BME) is a reducing agent and antioxidant that reduces the levels of oxygen radicals. It is usually added to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at 5% concentration. This is done because BME cleaves the intermolecular disulfide bonds and denatures proteins.
- To prepare base solvent add 3ml 20% SDS to add 3.75mL 1M Tris buffer at pH 6.8 in a suitable container.
- Add 9 mg bromphenol blue, 1.16 gm DTT (or 2.4ml B-mercaptoethanol) and mix well.
- Add 4.5mL glycerol to the solution, mix well.
Mercaptoethanol, 2-
2-Mercaptoethanol (2-ME) is a clear colorless to very faint yellow liquid that boils at 157–158 °C and has a concentration of 14.3 M (mol l−1).
- 50 mM Tris-HCl pH 6.8.
- 2% SDS.
- 10% glycerol.
- 1% β-mercaptoethanol.
- 12.5 mM EDTA.
- 0.02 % bromophenol blue.
NuPAGE LDS Sample Buffer (4X) is used to prepare protein samples for denaturing gel electrophoresis with Bis-Tris or Tris-Acetate gels. It contains lithium dodecyl sulfate, pH 8.4, which allows for maximum activity of the reducing agent.
Many protocols add b-mercaptoethanol to cell media (concentrations around 0.05 mM) in order to keep T cells, macrophages, and DC cultures viable; however, some protocols do not.
- 4% SDS.
- 20% glycerol.
- 0.004% bromphenol blue.
- 0.125M Tris-Cl, pH 6.8.
- 10% 2-mercaptoethanol (or DTT) (add immediately before use)
4x Laemmli sample buffer: Add 100 µl of 2-mercaptoethanol per 900 µl (final concentration of 355 mM). Alternatively, add dithiothreitol (DTT or Cleland's reagent) to a final concentration of 50 mM. Note: For best results, do not store sample buffer with 2-mercaptoethanol.
How do you make a 5x buffer?
- Dissolve in 700 ml of H2O: 15.1g Tris base. 94g glycine. 50ml of 10% SDS.
- After solid is dissolved, adjust volume to 1L with H2O.
It means how concentrated it is, ie how many times (hence the X) working concentration (or 1X) it is. So, for example, your 2X buffer is 2 times more concentrated than a working concentration of the buffer.
- 2.5 ml 1 M Tris-HCl pH 6.8.
- 0.5 ml of ddH20.
- 1.0 g SDS.
- 0.8 ml 0.1% Bromophenol Blue.
- 4 ml 100% glycerol.
- 2 ml 14.3 M β-mercaptoethanol (100% stock)
50 µl of diluted beta mercaptoethanol (dilute 5µl to 720 µl distilled water to get 0.1M-concentrated BME is 14.3 M-if we then take 50µl to 100ml we get 0.05 mM final concentration) 1 ml L-glutamine (Fisher MT-20-005-LI) 1 ml Na-Pyruvate (Fisher MT-20-000-LI from 6x100ml bottles)
Gibco™ 2-Mercaptoethanol (also known as beta-mercaptoethanol or BME) is a potent reducing agent used in cell culture media to prevent toxic levels of oxygen radicals. 2-Mercaptoethanol is not stable in solution, so most protocols require daily supplementation.
Sigma-Aldrich provide BME concentration 14.3 M (pure liquid), yet the openwetware.org protocol for SDS sample buffers gives a molarity of 14.7 M.
Even though there is no major difference between them, both are anionic detergents, LDS (Lithium dodecyl sulfate) is a better detergent in comparison to SDS (sodium dodecyl sulfate) if the protein is to be resolved at low temperature.
Sample buffers are Core Foundation objects that the system uses to move media sample data through the media pipeline. An instance of CMSampleBuffer contains zero or more compressed (or uncompressed) samples of a particular media type and contains one of the following: A CMBlockBuffer of one or more media samples.
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Guide to Western Blot Sample Preparation.
| Buffer | Composition |
|---|---|
| Tris-Triton X-100 | 10 mM Tris, pH 7.4 100 mM NaCl 1 mM EDTA 1 mM EGTA 1% Triton X-100 10% glycerol 0.1% SDS 0.5% deoxycholate |
This buffer contains SDS and is suitable for denaturing gel electrophoresis. Dilute 1:4 with sample before loading. Note: 5X Sample Buffer should be brought to room temperature prior to use. Mix well before loading gel.
How do you make a 5X Laemmli sample buffer?
- Add 1 ml of 1% bromophenol blue to 4 ml of 1.5 M Tris-Cl pH 6.8.
- Add 10ml of glycerol and mix.
- Add 2 g of SDS and mix (the SDS will take a few minutes to dissolve).
- Add 5 ml of β-mercaptoethanol and mix.
- Aliquot and store at -20°C.
Make 5X GLB: 5% SDS, 50% Glycerol, 0.1% Bromophenol Blue, 250 mM Tris-HCl, pH 6.8, and, add 5% BME before use. o 0.1% Bromophenol Blue: 0.04 g Bromophenol Blue from solid or solution. For what it's worth, I think that adding BME strictly before use is overkill in most cases.
5X sample buffer is more concentrated than 2X buffer. We always load 1X on a gel. - To prepare samples in 2X sample buffer, dilute to 1X using 1:1 ratio ( sample: sample buffer) - To prepare samples in 5X sample buffer, dilute to 1X using 4:1 ratio (sample: sample buffer)
How to make 1x TBE buffer. Add 100 mL 10x TBE stock solution to a 1 L Duran bottle. Add 900 mL MilliQ water. Mix the solution by shaking.
6X Universal / Glow Dyes
Dilute one part 6X Dye solution into five parts of sample solution to give a final concentration of 1X Dye solution. The sample is then ready to load to a gel.
Concentrated solutions can be expressed in terms of fold-concentrated. If a standard, final concentration is termed 1X (1 fold concentrated), a solution concentrated ten-fold is termed 10X.