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Tris-Glycine




COMPATIBLE TANKS

NuSep provides a range of cassettes to suit your needs - no need to replace your current tank!

NB cassettes (10cm wide X 8.5cm high) will fit in all Bio-Rad™ Mini-Protean™ tanks (including tetra and dodeca cells).

NN cassettes (10cm wide X 10cm high) will fit in the Invitrogen™ X-Cell Sure-Lock™ gel apparatus.

NG cassettes (10cm wide X 8.0cm high) fit in all other 10cm tanks:

•  VWR Tanks
•  GradiGel Mini 4 -Cell
•  EC 4- Cell
•  Hoefer Tall Mighty Small (SE 280)
•  Hoefer Mighty Small II (SE 260/SE 250)
•  Hoefer Mini VE
•  Daiichi Mini 2-Gel & 6-Gel
•  Owl Road Runner, Penguin
•  Invitrogen™ Novex® XCell I and II and SureLock™
•  Bio-Rad® Mini-PROTEAN® II & 3
•  Owl Single Sided Vertical System
•  Shelton IBI protein system

BUFFER FORMULATIONS

Running Buffers (10x)

Running Buffer Recipe

Tris-HEPES-SDS

Tris-Glycine-SDS

Tris-Tricine-SDS

Tris

121 g

29 g

61 g

HEPES

238 g

-

-

Glycine

-

144 g

-

Tricine 

-

-

9 g

SDS 

10 g

10 g

10 g

Deionised water

to 1000 mL

to 1000 mL

to 1000 mL


TruSep sample buffer (2x, 10 mL, no reducing agent)

Component

Amount

Tris

0.15 g

SDS

0.4 g

Bromophenol blule

0.003 g

Glycerol

4 mL

5 M Hydrochloric acid

0.25 mL

 

WESTERN BLOTTING PROTOCOL

 

Wet-Blotting

Semi-Dry Blotting

Voltage

40 V

120 min

Time

20 V

30 min

Recommended Buffer

NuSep transfer buffer*

NuSep transfer buffer*

Membrane

Nitrocellulose or PVDF

Nitrocellulose or PVDF

*Standard Tris-Glycine can be used. Protocol may need to be optimised. 

COMPATIBLE STAINS

All proteins stains are compatible with nUView gels, including:

•  Coomassie blue
•  Silver and copper stains
•  Fluorescent stains


COMPATIBLE RUNNING BUFFERS

A range of buffers can be used with nUView gels. Each buffer produces different migration patterns.

•  Tris-Glycine (available from NuSep)
•  Tris-HEPES (available from NuSep)
•  Tris-MES
•  Tris- MOPS 
•  Tris-Tricine: for low molecular weight separations.


FREQUENTLY ASKED QUESTIONS


Do you sell gradient gels? 

Yes.

•  8-16% for mid-range separations
•  4-20% for wide-range separations


Do I have to buy a new tank?

No. NuSep gels are compatible with most popular mini-gel tanks. 

Do they have a stacker gel?

All the gels have a 4% stacking gel.

Do they contain SDS?

No, the gels do not contain SDS (SDS is incorporated in the running buffer solution). The gels can be used for native runs. 

Can I use reducing agent?

NuSep gels and sample buffer do not contain reducing agents. They can be added to a sample prior to loading. Beta-mercaptoethanol and DTT are both suitable for use with our gels. See Running formulations. 

What is the gel thickness?

1mm

Can the gels be dried?

Yes. NuSep gels must be air dried. Use of vacuum drying results in cracking. NuSep gels can only be dried crack-free with NuSep Gel Drying Solution. All other drying solutions will cause cracking. A recommended gel drying protocol can be found on the NuSep website.

TROUBLE SHOOTING


Problem
: Distorted protein bands.

Cause: Air bubbles in the sample wells, or between gel and cassette, or at the bottom of the cassette.

Solution: Use a transfer pipette to displace the air bubbles from the sample wells.


Problem
: Streaking.

Cause: Poorly soluble or weakly charged particles (such as carbohydrates) in sample.

Solution:
1 Centrifuge sample.

2 Change pH of buffer.

3 Heat sample in the presence of SDS.

Problem: Bands difficult to distinguish.

Cause: Incorrect gel selection, sample overloading, insufficient cooling buffer.

Solution:  

1 Reduce sample size.

2 Select a gel which separates in the desired molecular weight range.

3 For proteins of similar molecular weight a 2-D separation may be required.

4 Increase buffer in outer tank.

Problem: Sample spreading across the gel.

Cause: Too much salt in the sample.

Solution: Reduce salt by dialysis or ultra-filtration.

Problem: Sample contains appreciable carbohydrate.

Solution: Enzymatically remove the carbohydrate.

Problem: Sample contains lipoproteins.

Solution: Use a gel with large pore size at top. Try addition of a non-ionic detergent.

Problem: Protein denaturation and band inversion.

Cause: Excessive heating.

Solution: Always start with chilled buffer (<15°C).

Problem: Diffuse proteins zones in gel after staining.

Cause: SDS still present in gel.

Solution: Fix in 10% TCA prior to staining, otherwise use 30% methanol in the destaining solution.