Recommendations/Tips for Gel Running
1. Remove comb and tape before adaption. 2. Use fresh 1X running buffer for the inner cathode chamber. 3. Do not use Tris-Glycine running buffer for Q-PAGE™ Bis-Tris Precast Gels. 4. Rinse the wells before sample loading.
Sample Preparation for SDS-PAGE
1. Mix protein sample with 2X sample buffer.
2. Heat the diluted samples at 95°C for 5 min or at 70°C for 10 min.
3. Cool the diluted samples to 4°C and spin down the water condensed on tube surface. (If there is high viscosity part at bottom of tube, transfer supernatant to a new tube.)
Prepare Q-PAGE™ for Sample Loading
1.Open the blister tray of Q-PAGE™ Precast Gel.
2.Briefly rinse the gel cassette with ddH2O.
3.Remove tape and comb; avoid squeezing the gel.
4.Adapt Q-PAGE™ to electrophoresis system; instruction are provided below. (BioRad Mini-PROTEAN® Core Electrophoresis System is recommended.)
5.Use a pipette to gently wash the wells with running buffer to remove residual storage buffer.
6.Fill the wells with running buffer prior to sample loading.
7.Load samples and pre-stained protein marker into numbered wells.
8.Fill both inner and outer chambers with running buffer to the highest level. Ensure gel wells are completely covered.
Power Setting for Running Q-PAGE™
Optimize the voltage and running time if needed.
| 130 V | 180 V | 230 V*2 |
Running Time*1 | 45-60 mins | 25-40 mins | 15-30 mins |
Expected Current Initial (per gel) Final (per gel) | 60-70 mA 20-25 mA | 100-110 mA 40-50 mA | 130-140 mA 60-70 mA |
Expected temperature | 25-30°C | 25-35 °C | 35-45°C |
*1 Set voltage higher than 100 V is recommended.
*2 For higher voltage conditions, please use fresh running buffer for inner and outer chambers.
*3 Running time varies depending on gel percentage, running buffer, temperature, and power supply
Remove Q-PAGE™ Gel from Cassette
Open cassette immediately after electrophoresis. Avoid gel drying.
1.Insert the cassette opener into corners of cassette.
2.Sequentially pry the opener to separate the two plates.
3.Gently pull two plates apart from the top of cassette.
4.Carefully detach the gel either from the bottom of gel or the top side of the cassette.
-Avoid diagonally peeling the gel from the corner.
-Use water to help gel detachment if it needed
5.Gently remove the gel for further staining or Western blotting.
Gel Staining Proteins separated using Q-PAGE™ Precast Gels can be further stained with most popular staining reagents, such as Coomassie dyes (R-250 or G-250), Silver-stain solution, and FluoroStain™ Protein Fluorescent Staining Dye. (Cat. No. PS1000)
Transferring Protein from Q-PAGE™ to Blotting Membrane 1. After protein separation using Q-PAGE™, gently detach QPAGE™ from cassette and then equilibrate the gel in transfer buffer. 2. Pre-soak blotting membrane and filter papers in transfer buffer. 3. Assemble transfer sandwich by orientating cathode, sponge, filter papers, gel, membrane, filter papers, sponge, and anode. The protein goes to the direction of cathode to anode. 4. Carefully move roller over the gel/membrane to remove air bubbles and excess buffer until complete contact is established. 5. Insert transfer cassette into transfer module. Notice that black side of cassette should be next to black side of module. 6. Fill transfer tank with pre-cooled transfer buffer to the highest water level. 7. Set constant voltage at 100 V. Transfer for 90 minutes at low temperature condition. Pre-stained protein marker should be visible on the membrane after transfer is completed. Transfer of proteins to the membrane can be checked using Ponceau S staining before blocking step.
Supplemental Information for Using Q-PAGE™ Precast Gel
Adapting Q-PAGE™ Mini Precast Gel to BioRad Mini-PROTEAN® Core 1. After removing comb and tape, place the Q-PAGE™ Mini Precast Gel with notched plate facing toward inner chamber. 2. Align the notched plate to ensure the edge sits just below the notch at the top of green gasket. 3. Gently press gel cassette toward green gasket and then lock gel cassette with two green arms. Avoid squeezing the cassette and gel.
4. Fill inner chamber with running buffer to check tightness of seal. If necessary, reassemble and check the seal again. 5. Fill inner chamber with running buffer to ensure gel wells are completely covered. 6. Fill outer chamber with running buffer to the highest level.
Adapting Q-PAGE™ Mini Precast Gels to other electrophoresis system, please follow the manufacturer’s instruction.
Buffer recipes
2X sample buffer with reducing agent 62.5 mM Tris-HCl pH 6.8, 2% SDS, 25% (v/v) glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol or 100 mM DTT (added fresh)
10X MOPS running buffer 60.6 g Tris base, 104.6 g MOPS, 10.0 g SDS, 3.0 g EDTA. Bring up the volume to 1 L with ddH2O.
10X MES running buffer 60.6 g Tris base, 97.6 g MES, 10.0 g SDS, 3.0 g EDTA. Bring up the volume to 1 L with ddH2O.
1X running buffer Dilute 100 ml 10X running buffer with 900 ml ddH2O.
10X transfer buffer 30.0 g Tris base, 144.0 g Glycine. Bring up the volume to 1 L with ddH2O.
1X transfer buffer *Cool 1X transfer buffer to 4°C before using. Dilute 100 ml 10X transfer buffer with 200 ml methanol and 700 ml ddH2O. **Add SDS to 0.1% to promote transfer of high molecular weight proteins.
