Product Information
This kit provides a simple and rapid preparation ofone-step PAGE coloured gels,suitable for Tris-glycine electrophoresis system, can be used for SDS-PAGE gel electrophoresis and non-denaturing PAGE gel electrophoresis.
Compared with traditionalacrylamide kits, there are the following advantages：
One-step cast, easy to use, fast and efficient: The kit is designed as a premixedsolution for thestacking andresolving gel, which cancast gel by adding themodified coagulant. Aftercasting theresolving gel, there is no need for liquid sealing to flatten the gel surface and wait for gelation, and thestaking gel can becasted directly..
No need to add TEMED to avoid odours:No need to add TEMEDduring casting,thusavoid exposure to odourous and toxic reagents.
Colourfulstacking gel with clear indications:The kit contains two stacking gel dyes (red/green) for the preparation of different coloured gels with clearly visible wells and for the differentiation of gels containing different samples.Thecolorfuldye is stable in thestacking gel and does not migrate to theresolving gel with electrophoresis, thus not affecting the electrophoresis and staining effect. It is also easy to identify and excise thestacking gel after electrophoresis is completed, without affecting subsequent experiments such as Western Blot.
Excellent separation effect: protein bands after electrophoresis are flat, clear, delicate and sharp, especially for small molecule proteins.
The One-StepFastcast Colorful AcrylamideKits range consists of gel kits with different concentrations, including 6%, 8%, 10%, 12% and 15%, for the separation of proteins of different molecular weights. The kits are supplied with two colours ofstacking gel dye andmodified coagulant. Approximately 50 regular sized gels can be prepared, depending on the thickness and size of the gel. See below for a list of product numbers.
This manual applies to the One-StepFastcast Colorful AcrylamideKit series (G2175, G2176, G2177, G2178, G2179)..
- Product Number of this series
|
Cat.No. |
Concentration of gel |
Quantity of gel that can be casted (approx.) |
|
G2175 |
6% |
60 pieces of 0.75 mm gel, or 50 pieces of 1.0 mm gel, or 30 pieces of 1.5 mm gel |
|
G2176 |
8% |
|
|
G2177 |
10% |
|
|
G2178 |
12% |
|
|
G2179 |
15% |
- List of kits
|
component name |
Spec. |
|
StackerA |
50 mL |
|
StackerB |
50 mL |
|
Resolver A |
125 mL |
|
Resolver B |
125 mL |
|
Polyacrylamidegel (PAGE)stacker reddye(500×) |
100 μL |
|
Polyacrylamidegel (PAGE)stacker green dye(500×) |
100 μL |
|
Modified coagulant |
5×1 mL |
Storage and Shipping Conditions
Ship with wet ice;Modified coagulant promoterstore at-20 ℃, otherstore at2-8 ℃ away from light,valid for 12 months.
Assay Protocol
1. Choose appropriate concentration of acrylamide solution according to molecular weight of targetprotein:
|
Concentration of acrylamide |
Best separation range (kDa) (Tris-Glycine Buffer, G2018) |
Best separation range (kDa) (SWE Fast High-Resolution Electrophoresis Buffer, G2081) |
|
6% |
50-300 |
15-300 |
|
8% |
30-130 |
10-250 |
|
10% |
20-100 |
5-150 |
|
12% |
10-60 |
3-100 |
|
15% |
<40 |
<60 |
2. Follow the steps below to cast the gel. Note that the amount of reagents used in each step and the amount of gel volume required for different thicknesses of glass plates are shown in the table below.
(1) Take equal volumes ofResolver A andResolver B and mix well
(2) Take equal volumes ofStacker A andStacker B, then accurately aspirate the Polyacrylamidegel (PAGE)stacker dye, add to the gel solution and mix well.Note: The Polyacrylamidegel (PAGE)stacker dye is easy to settle, and should be mixed well before use. In addition, thestacker dye can alsonot be added, then thestacking gel is colourless.
(3) Add the modifiedcoagulant to theresolving gel mixture solution of (1) and mix gently. Inject the mixed gel solution into the assembledhandcast glass plate so that the distance between the liquid surface and the upper edge of the concave glass plate is 0.5 cm longer than the length of the comb teeth
Note:When mixing after adding themodified coagulant, the action should be gentle to prevent a large number of air bubbles produced by violent mixing. The volume of theresolving gel solution already contains the appropriate amount of redundancy, please do not use all forcastingtheresolving gel, in order to avoid thestacking gel height is not enough.
(4) Add the modified coagulant to thestacking gelmixture solution of (2) and mix gently.Without waiting for the resolving gelto solidify, the mixedstacking gel solution is directly injected into thehandcast glass plate, and then insert the comb teeth.Note:When mixing after adding themodified coagulant, the action should be gentle to prevent a large number of air bubbles produced by violent mixing. And try to be as gentle as possible when injecting thestacking gel, to avoid rushing thestacking gel solution into theresolving gel.
(5) After about 10-15 min, the gel is solidified, and the electrophoresis can be carried out after removing the teeth of the comb. Note: It is normal for thestacking andresolving gel to be slightly uneven at the demarcation line after gel solidification, which will not affect the subsequent electrophoresis.
The volume of thestacking andresolving gel solutions can be adjusted proportionally for glass plates of different sizes and thicknesses. Taking the commonly used 8.3 cm x 7.3 cm gel plate as an example, the recommended preparation system is shown in the following table:
|
Preparation group |
Component |
0.75 mm Glass Plates |
1.0 mm Glass Plates |
1.5 mm Glass Plates |
|
resolving gel solution |
Stacker A |
2 mL |
2.5 mL |
4 mL |
|
Stacker B |
2 mL |
2.5 mL |
4 mL |
|
|
Modified coagulant |
24 μL |
30 μL |
48 μL |
|
|
stacking gel solution |
Resolver A |
1 mL |
1 mL |
1.5 mL |
|
Resolver B |
1 mL |
1 mL |
1.5 mL |
|
|
Modified coagulant |
12 μL |
12 μL |
18 μL |
|
|
Stacker dye (red or green) |
4 μL |
4 μL |
6 μL |
3. Recommended electrophoresis conditions:
a) Electrophoresis with SWE Rapid High Resolution Electrophoresis Buffer (G2081): 150-250 V, 25-50 min;
b) Or use Tris-Glycine electrophoresis buffer (G2018) for electrophoresis:stacking gel 90 V for about 30 min (marker goes intoresolving gel);resolving gel 150-180 V for about 60-90 min (can be adjusted accordingly).
Note
1. Whencasting thestacking gel needs to be gently and left-right panning, to avoid focusing on a position tocast, so as to avoid thestacking gel solution into theresolving gel to destroy the flatness of thestacking andresolving gel demarcation line,casting thestacking gel need to use a 1mL pipette, do not pour vertically, to minimize the impact force.
2. The Polyacrylamidegel (PAGE)stacker dye (green or red) is easy to settle, so please blow lightly to mix before use.
3. If the temperature of the preparedStacker/Resolver premix is low (just taken out of the refrigerator), please return it to room temperature before proceeding with the subsequent operations, to avoid prolonged gelation time due to its low temperature.
4. If thecasted gel cannot be used on the same day, it can be stored at 4℃ for 1-2 days.
5. Modified coagulant is more stable than ammonium persulphate (AP),for routine use, it canstore at 4℃ for six months; if not used for a long time, please place at -20 ℃ to avoid repeated freezing and thawing.
6. The presence of acrylamide in the premixed liquid is harmful to human, please pay attention to protective measures during operation.
7. Cast the gel as soon as possible after adding the modified coagulant, do not leave it for a long time.
8. There is a significant positive correlation between gelation speed and temperature. Under the same conditions, the higher the temperature, the faster the gelation speed, room temperature is too high, it is recommended to reduce the amount of modified coagulant promoter; on the contrary, if the room temperature is low, it can be appropriate to extend the gelation time.
9. If you need to accelerate the speed of gelation, you can add the appropriate amount of TEMED beforecasting, and at the same time increase the amount of modified coagulant by 0.5 times.
10. For your safety and health, please wear a lab coat and disposable gloves.
For Research Use Only!
