What is NuPAGE?
Invitrogen NuPAGE Bis-Tris protein gels are precast polyacrylamide gels that provide broad molecular weight protein separation with high resolution and sample integrity. These precast gels are ideal for applications where protein integrity is crucial.
What is MES Running buffer?
NuPAGE MES SDS Running Buffer (20X) is formulated for running proteins on NuPAGE Bis-Tris gels. It is recommended for separating small- to medium-sized proteins.
Can you reuse MOPS buffer?
Sometimes, if the buffer goes bad, the gel runs crooked. But this can also happen if the buffer level is low. In this case, if you re-add fresh running buffer, the gel starts running properly again. So, the bottom line is: You can safely re-use the running buffer upto three times.
What is NuPAGE antioxidant?
NuPAGE Antioxidant prevents sample reoxidation and maintains proteins in a reduced state during protein gel electrophoresis and protein transfer. It is added to the running buffer when performing protein gel electrophoresis under reducing conditions.
What is in NuPAGE transfer buffer?
(20X) NuPAGE transfer buffer (500 ml) Bicine 40.8 g (500 mM) Bis-Tris 52.32 g (500 mM) EDTA free acid 3.0 g (20.5 mM) Ultrapure water to 500 ml. 1X buffer should be pH 7.2 (do not adjust with acid or base).
Can you autoclave MES buffer?
Autoclaving is not recommended for any sulfonic acid buffer. When MES solutions are autoclaved, they turn yellow (although pH does not change measurably). The identity of the yellow breakdown product is unknown. When MES solutions are stored at room temperature, they can turn yellow within six months.
How do you make a MES buffer?
MES (0.5 M, pH 6) Preparation and Recipe
- Prepare 800 mL of dH2O in a suitable container.
- Add 97.62 g of MES free acid to the solution.
- Starting pH for 0.5M MES solution is 3.23 For 1 L of 0.5 M MES, 13.6 ml of 10N NaOH is needed to adjust the pH to 6.0.
- Add dH2O until the volume is 1 L.
How long can gel sit before transfer?
It is preferable that the transferring of protein bands exactly after the running of them to prevent diffusion of proteins specially in non purified proteins. Although, you can keep the gel up to 12 hours at 4◦ C. Of course, store the membrane in TBS-T. Never leave the membrane dry!
How do you make MOPS buffer?
MOPS Buffer (10X) (0.2 M, pH 7) Preparation and Recipe
- Prepare 800 mL of dH2O in a suitable container.
- Add 41.86 g of MOPS free acid to the solution.
- Add 4.1 g of Sodium Acetate to the solution.
- Add 3.72 g of Disodium EDTA to the solution.
- Adjust solution to desired pH using NaOH (typical pH = 7)
What is the pH of hepes?
6.8 to 8.2
HEPES has the following characteristics: pKa1 (25 °C) = 3. pKa2 (25 °C) = 7.5. Useful pH range = 2.5 to 3.5 or 6.8 to 8.2.
Why is Western blotting called immunoblotting?
A western blot experiment, or western blotting (also called immunoblotting, because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis.
Why do we use methanol in transfer buffer?
The presence of methanol in the transfer buffer serves two main purposes: It promotes dissociation of SDS from the protein and dramatically improves adsorption of proteins onto membranes in the presence of SDS, although these effects may vary with proteins.
How do I make Bis Tris gels?
How to Make Your Own Bis-Tris Gels
- 5X Low MW Running Buffer.
- 5X High MW Running Buffer. 250 mM MOPS. 250 mM Tris.
- 200X Running Buffer Reducing Agent. 1 M sodium bisulfite. Add to running buffer at 5mM final concentration.
- 3.5X Gel Buffer. 1.25 M bis-Tris (pH 6.5-6.8 with HCl)
Why does MES buffer turn yellow?
The yellow color is the result of aging but does not cause a change in pH. Autoclaving the buffer produces the yellow color immediately.
How do you adjust the pH of MES?
What type of acid is MES?
MES is the common name for the compound 2-(N-morpholino)ethanesulfonic acid. Its chemical structure contains a morpholine ring. It has a molecular weight of 195.2 and the chemical formula is C6H13NO4S.
Can I autoclave MES buffer?
Can you run a gel for too long?
If you run gel electrophoresis too long, the sample can run out of the bottom of the gel.
What happens if you run your gel too long?
However, if the electrophoresis is conducted for too long, DNA bands may migrate off the end of the gel. The higher the voltage, the faster the DNA will travel through the gel. However, voltages that are too high can possibly melt the gel or cause smearing or distortion of DNA bands.
Can you leave a gel overnight?
Agarose gel has a storage life of about 3 – 4 weeks if it is mixed with specified amount of buffer solution and it should be stored in dark at a temperature of around 4 0C. It is very light sensitive and should not be kept under light for more than 3 hours.
What is MOPS buffer made of?
MOPS is the common name for the buffering compound in MOPS buffer. MOPS stands for 3-(N-morpholino) propanesulfonic acid, and with a pKa of 7.20, makes a good buffering agent for many biological systems requiring neutral pH. HEPES is a chemically similar pH buffering compound.
What is the pH of MOPS buffer?
SOLUBILITY / SOLUTION STABILITY: MOPS Sodium is very soluble in water, at least to 33% (w/w), giving a clear colorless solution. The pH of a 0.1 M solution is generally 10-12 (temperature-dependent).
How to use nupage Mes SDS running buffer (np0002)?
Use NuPAGE Antioxidant (NP0005) in the running buffer to maintain the reduced state of the proteins during the run and to allow maximum band sharpness. The gels can be run using NuPAGE MES SDS Running Buffer (NP0002) to better resolve small proteins or NuPAGE MOPS SDS Running Buffer (NP000102) to resolve medium- to large-size proteins.
How do I use nupage Bis-tris gels for gel electrophoresis?
NuPAGE Bis-Tris gels are formulated for denaturing gel electrophoresis applications. For optimal sample preparation, use the NuPAGE LDS Sample Buffer and NuPAGE Sample Reducing Agent. Use NuPAGE Antioxidant in the running buffer to maintain the reduced state of the proteins during the run and to allow maximum band sharpness.
What is the difference between nupage Bis-Tris and Tris-glycine gels?
Unlike traditional Tris-glycine gels, NuPAGE Bis-Tris gels have a neutral pH environment that minimizes protein modifications. Use NuPAGE Bis-Tris gels for preparing proteins for sequencing, mass spectrometry, and any other techniques where protein integrity is crucial. Also, use NuPAGE gels for optimal results during day-to-day use.
What gels are compatible with the mini gel tank?
The Mini Gel Tank is compatible with all Invitrogen Novex, NuPAGE, and Bolt mini gels. Each Mini Gel Tank can accommodate up to two gels per run. The unique tank design enables convenient, side-by-side gel loading and enhanced viewing during use.