3.1 Bacmid generation for human GS-GN complex
3.1.1 Bacmid transformation
Fig. 2 shows the GYS1 and GYG1 genes, encoding for GS and GN respectively, in constructs suitable for protein production in insect cells. pFL (Fig. 2a) is a bicistronic vector encoding both genes and can be used as a single infection method. pFastBac HT B (Fig. 2b) encodes each gene in a separate vector and can be used as a co-infection method. Both methods produce active, stoichiometric complexes, however the pFastBac HTB co-infection protocol has been optimised to balance differences in expression between GS and GN and avoid high excess GN protein during purification. If expression of mutant complexes is required, we recommend this co-expression method as this will reduce cloning efforts.
1. Thaw 1 x 100 µL DH10MultiBac Turbo electrocompetent cells per construct on ice.
2. Quantify plasmid DNA using a nanodrop or similar spectrophotometric technique and add 500-1000 ng of plasmid DNA to the cells, under sterile conditions (i.e. next to a flame).
3. Incubate on ice for 30 minutes.
4. Transfer the mixture to a chilled electroporation cuvette (Bio-Rad GenePulser).
5. Insert the cuvette into a MicroPulser™ machine (Bio-Rad).
6. Electroporate using the preprogramed bacterial setting (1.8 kV, 1 pulse). If using pFastBac constructs, chemically competent cells can also be used (see Note 8).
7. Incubate on ice for 5-10 minutes.
8. Add 450 µL sterile LB.
9. Incubate at 37oC shaking at 200 rpm for 4-6 hours.
10. For each construct, prepare 5 LB-Agar plates (20 mL per plate) containing 10 µg/mL gentamycin, 50 µg/mL kanamycin, 10 µg/mL tetracycline, 100 µg/mL ampicillin and 40 µg/mL IPTG. Store the plates protected from light (see Note 9).
11. Save 1 plate per construct at 4oC protected from light to be used later. An hour before the transformation mix has finished the incubation period, spread 100 µL of 16.6 mg/mL X-gal onto each plate and leave to dry at 37oC, protected from light.
12. After the required incubation time for the transformation mix, prepare serial dilutions of the transformed cells:
To 50 µL transformation mix, add 450 µL LB = 10-1
To 50 µL of 10-1, add 450 µL LB = 10-2
To 50 µL of 10-2, add 450 µL LB = 10-3
13. Spread 100 µL of neat, 10-1, 10-2 and 10-3 dilutions of transformed cells onto separate plates.
14. Incubate at 37oC for 48 hours, protected from light.
3.1.2 Blue-white colony screening
1. After 48 hours, check all plates for white colonies.
2. Add100 µL 16.6 mg/mL X-gal to the 5th plate (stored at 4oC, as described in point 11 of the “Bacmid transformation” section) and leave to dry at 37oC, protected from light.
3. Prepare 5 x 5 mL tubes with LB containing 50 µg/mL kanamycin, 10 µg/mL tetracycline, 10 µg/mL gentamycin and 100 µg/mL ampicillin.
4. If possible, choose 3-5 white colonies per construct. Re-streak each colony on to the LB-agar plate and also set up a 5 mL overnight culture with the same colony.
5. Re-streak one blue colony onto the plate as a negative control.
6. Incubate plate overnight at37oC.
7. Incubate cultures overnight at 37oC shaking at 200 rpm.
3.1.3 Bacmid DNA purification
1. After overnight incubations, check which colonies have a confirmed white phenotype on the re-streaked plate.
2. For the corresponding overnight growth that you wish to purify, harvest cells by centrifugation at 2,900 g for 10 minutes at 4oC.
3. Discard the supernatant.
4. For the following steps, use Qiagen miniprep buffers: P1, P2, N3.
5. Resuspend the pellet in 300 µL P1 buffer.
6. Add 300 µL P2 buffer, invert to mix gently and incubate for no longer than 5 minutes.
7. Add 300 µL N3 buffer and invert and mix until homogenous.
8. Spin mixture at 17,000 g for 12 minutes at room temperature.
9. Transfer supernatant to a new tube.
10. Spin at 17,000 g for 8 minutes at room temperature.
11. Transfer supernatant to a new tube.
12. Add 700 µL 100% isopropanol and invert slowly to mix.
13. Spin at 17,000 g for 12 minutes at room temperature.
14. Without disturbing the pellet, remove the isopropanol.
15. Carefully add 200 µL 70% ethanol.
16. Spin at 17,000 g for 8 minutes at room temperature.
17. Carefully remove ethanol.
18. Add 50 µL of 70% ethanol carefully and without disturbing the pellet (aim for the opposite side of pellet), for sterilitiy.
19. Move to a cell culture hood.
20. Remove the ethanol without disturbing the pellet.
21. Dry for 10 minutes (or longer if necessary) with the tube lid open.
22. Add 35 µL filtered H2O, close tube and tap gently. Mix and let the pellet dissolve for 10 minutes.
23. Take a 5 µL aliquot into a fresh tube. Keep the original tube in sterile conditions (i.e. do not open unless in a cell culture hood).
24. Measure the concentration using a nanodrop. A typical concentration should be between 800-1500 ng/µL.
3.2 Culturing and transfecting insect cells
3.2.1 Transfection to generate a P1 virus
To express protein in insect cells, Sf9 cells first need to be transfected with a recombinant bacmid DNA construct (bacmid generated as described in sections 3.1.1-3.1.3). This generates a P1 virus.
1. Dilute Sf9 cells to a density of 1.0 x 106 cells/mL using Sf-900™ II SFM media containing L-glutamine but no antibiotic-antimycotic to the volume required.
2. Seed 5 mL of cells into T25 flasks. Set up two flasks per transfection: one for the transfection, and one for a mock transfection control.
3. Incubate for 30 minutes at 27oC in a static incubator so that cells can attach to the surface.
4. Dilute each bacmid DNA to 250 ng/µL using filter sterilised H2O.
5. Prepare two tubes for each bacmid DNA that needs to be transfected. In one tube, add 30 µL of bacmid DNA at 250 ng/µL to 300 µL Sf-900™ II SFM media containing L-glutamine but no antibiotic-antimycotic.
6. For the mock, add 30 µL of filter sterilised H2O instead of bacmid DNA.
7. In a different tube, add 10 µL of transfection reagent to 100 µL of Sf-900™ II SFM media containing L-glutamine but no antibiotic-antimycotic.
8. Add the DNA mix to the transfection reagent mix.
9. Incubate at room temperature for 30 minutes.
10. After the cells have been incubated for 30 minutes, wash the cells three times with 5 mL of Sf-900™ II SFM media containing L-glutamine but no antibiotic-antimycotic.
11. After the DNA/transfection reagent mix has incubated for 30 minutes, add each 440 µL reaction to the corresponding flask.
12. Incubate for 24 hours in a static incubator at 27oC.
13. After 24 hours, remove the media in the T25 flasks.
14. Add 6 mL Sf-900™ II SFM media supplemented with L-glutamine and antibiotic-antimycotic to the T25 flasks.
15. Incubate for a further 6 days in a static incubator at 27 oC.
16. Visualise the cells. Compare the transfected cells to the mock-transfected cells. Transfected cells should appear larger, irregular or less circular in shape and more cells should be floating (Fig. 3).
17. If DH10MultiBac-YFP Turbo cells were used, also visualise YFP levels (in this case, using a GFP filter). High levels of YFP indicate a successful transfection (Fig. 3b).
18. Transfer the supernatant to a 15 mL falcon tube.
19. Harvest P1 virus by centrifugation at 1,000 g for 5 minutes at 4oC.
20. Transfer the supernatant to a new falcon tube. This is your P1 virus. Store at 4ºC protected from light. For long term storage, snap freeze with liquid nitrogen and store at -80ºC.
3.2.2 Generating P2 virus
Following generation of a P1 virus (as described in section 3.1.4), a high titer P2 virus needs to be produced to allow successful protein expression.
1. Dilute Sf9 cells to a density of 1.5 x 106 cells/mL using Sf-900™ II SFM media supplemented with L-glutamine and antibiotic-antimycotic, into a 1 L shake flask.
2. Add P1 virus to these cells, at a ratio of 1 mL P1:300 mL cells (Fig. 2).
3. Incubate at 27oC shaking at 120 rpm for 72 hours.
4. After 72 hours, visualise cells for signs of infection. Cells should appear bigger in size and irregular or elongated in shape. Cell growth should have remained static or cells reduced in number. Observation of dead cells and cell debris is normal. If cells don’t look infected, leave for an extra day.
5. Harvest by centrifugation at 1,000 g for 5 minutes at 4oC.
6. Transfer the supernatant to new falcon tubes. This is your P2 virus. Store at 4oC protected from light.
3.2.3 Growth for protein production
For protein expression, the P2 virus generated (see section 3.1.5) is used to infect either Sf9 or Tni cells.
1. Dilute cells to achieve a final density of 2.0 x 106 cells/mL using the appropriate media (supplemented with antibiotic-antimycotic with or without L-glutamine) (see section 2.1).
2. If using the bicistronic single vector pFL (Fig. 2a), use P2 virus to inoculate cells in a 1:10 ratio for virus:total growth culture (e.g. add 60 mL P2 virus to 540 mL cells) in a shake flask.
3. If using the co-infection method (Fig. 2b) use nine parts GS virus to one part GN virus (e.g. add 108 mL GS P2 virus and 12 mL GN P2 virus (total of 120 mL virus) to 480 mL cells) in a shake flask.
4. Incubate at 27oC shaking at 120 rpm for 48 hours.
5. After 48 hours, visualise cells for signs of infection. Cells should appear bigger in size and irregular or elongated in shape. Cell growth should have remained static or cells reduced in number. Observation of dead cells and cell debris is normal. If cells don’t look infected, leave for an extra day.
6. Harvest by centrifugation at 500 g for 15 minutes at 4oC with slow acceleration and deacceleration rates (typically a “4” setting in a 1-9 scale).
7. Discard the supernatant.
8. Wash Sf9 cells pellets two/three times with cold 1X PBS. These washing steps are not required if Tni cells are used for protein expression.
9. Snap freeze in LN2 and store at -80 ºC.
3.3 Purification of human GS-GN complex
This section describes the purification of the GS-GN complex. Here, the GN self-glucosylating tyrosine residue, 195 in humans, is mutated to a phenylalanine (Y195F) to produce a non-glucosylated GN species (12, 13). All reagents and purification samples are kept on ice or 4oC cold room or chiller cabinet unless stated otherwise.
3.3.1 Lysate preparation
1. Make lysis buffer by adding 0.8 mL of 200 mM PMSF and 2 mL of 30 mg/mL lysozyme to 150 mL of 1X low salt buffer. Make up to 200 mL using 1X low salt buffer to create final concentrations of 0.8 mM PMSF and 0.3 mg/mL lysozyme.
2. Re-suspend pellet in ~100 mL lysis buffer.
3. Lyse cells by sonication on ice, with the program: 5 minutes, pulse: 1 second on, 3 seconds off, 40% amplitude, temperature 4oC.
4. Take 30 µL sample after sonication (total lysate sample, TL).
5. Pellet insoluble material by centrifugation at 35,000 g, 30 minutes, 4oC.
6. Remove the supernatant, this is the soluble fraction.
7. Sonicate the soluble fraction on ice, to sheer DNA, using the program: 1 minute, pulse: 1 second on, 3 seconds off, 40% amplitude at 4oC.
8. Filter lysate using a 0.45 µM syringe filter.
9. Take 30 µL sample of insoluble fraction and filtered lysate. For the insoluble fraction sample, scrape the insoluble material using a pipette tip and add to 100 µL lysis buffer. Remove the pipette tip and vortex, then take 30 µL sample.
10. Continue to nickel affinity chromatography; freezing at this stage is not recommended.
3.3.2 Nickel affinity chromatography
1. Use a syringe or peristaltic pump to wash a 5 mL HisTrap HP column with 20 mL (4 column volumes) filtered H2O.
2. Equilibrate the same column with 20 mL 1X low salt buffer.
3. Load filtered lysate onto HisTrap column using peristaltic pump at 4oC and collect the flow through (FT).
4. Wash the column as follows and collect the washes: 20 mL 1X low salt buffer (W1), 20 mL 1X high salt buffer (W2), 20 mL 1X low salt buffer (W3).
5. Make sure the ÄKTA has been washed with filtered H2O and equilibrated with 1X low salt.
6. Attach HisTrap column to the ÄKTA. Run a program with the parameters at 2.5 mL/min collecting 5 mL fractions. Beginning with a wash of 30 mL of 1X low salt buffer, then run a gradient elution from 0-100% buffer B (elution buffer) across 150 mL, followed by an equilibration of 15 mL with 1X low salt buffer.
7. Run an SDS-PAGE gel of the lysate preparation samples (see Note 10), and fractions from the chromatogram (Fig. 4). All samples are 30 µL with the addition of 10 µL 4X Laemmli buffer, boiled for 10 minutes at 95 oC. Refer to section 3.4 if a step-by-step method for running an SDS-PAGE gel is required. Keep fractions on ice or at 4 °C while running gel.
8. Pool the fractions containing desired protein.
9. Measure A280 (e.g. using a nanodrop) and calculate the concentration using the extinction coefficient (ε=1.44) and Beer-Lambert’s law equation (A=εcl), where l is the path length, and A is the absorbance).
10. Take 30 µL sample (before dialysis sample).
11. Add TEV protease to protein (1 mg of TEV protease per 50 mg of protein).
12. Add the protein and His-tagged TEV protease to 10K MWCO dialysis tubing, and dialyse overnight at 4oC in dialysis buffer, stirring gently.
3.3.3 Nickel subtraction
Following lysate preparation and nickel affinity chromatography (see sections 3.2.1 and 3.2.2), a nickel subtraction step is performed to remove the His-tagged TEV protease and any remaining His-tagged GS-GN from the cleaved GS-GN complex.
1. Collect the dialysed protein and measure A280.
2. Take 30 µL sample (dialysed protein sample).
3. Wash the same HisTrap column with 20 mL filtered H2O and equilibrate with 20 mL 1X low salt buffer.
4. Load dialysed protein onto HisTrap column using peristaltic pump.
5. Collect flow through, this is your protein of interest (cleaved protein).
6. Wash the column as follows and collect the washes: 20 mL 1X low salt buffer (W1), 20 mL 1X high salt buffer (W2), 20 mL 1X low salt buffer (W3).
7. Run elution buffer through the same HisTrap column: 20 mL elution buffer (E1), 20 mL of elution buffer (E2). This will elute any remaining His-tagged protein bound to the column. This can be re-dialysed again with TEV to cleave the His tag.
8. Take 30 µL sample of flow through, W1, W2, W3, E1 and E2.
9. Measure A280 of flow through, W1, W2, W3, E1 and E2.
10. Run an SDS-PAGE gel (Fig. 5). Keep samples on ice or at 4 °C while running gel.
3.3.4 Size exclusion chromatography
1. Pool samples containing pure, cleaved protein.
2. Concentrate the protein to a suitable volume to load onto a size exclusion chromatography column, depending on the size of the injection loop to be used, using a Vivaspin 30,000 MW cut-off concentrator.
3. Measure A280 and calculate concentration.
4. Spin concentrated protein at 17,000 g for 15 minutes at 4oC to remove any precipitated protein.
5. Transfer the supernatant to a new 1.5 mL tube.
6. Take 5 µL sample (load sample) to run on gel. Add 25 µL of buffer to this sample, then add 10 µL 4X Laemmli buffer before running on a gel.
7. Measure A280 and calculate the concentration.
8. Inject onto a Superdex S200 16/600 column equilibrated in size exclusion buffer. Run a program with sample application from the capillary loop, emptying the loop with 3 mL. Then run an elution of 130 mL at a speed of 0.8 mL/min, beginning fractionation after 20 mL and collecting 1.4 mL fractions.
9. Run the load sample and the appropriate fractions from the size exclusion chromatogram on an SDS-PAGE gel (Fig. 6).
10. Pool and concentrate complex-containing fractions using a Vivaspin 30,000 MW cut-off concentrator.
11. Concentrate to desired concentration (usually 2-3 mg/mL), measuring A280 throughout this process.
12. Make 10 µL aliquots in PCR tubes and flash freeze in liquid nitrogen before storing at -80oC. Thaw aliquots in your hand by holding the PCR tube tight (minimising thawing time), before use.
3.4 Differential scanning fluorimetry
Differential scanning fluorimetry (DSF) is an assay that measures the thermal stability of a target protein. With the use of a hydrophobic fluorescent dye, changes in fluorescence can be monitored over a range of temperatures, and thus a melting temperature (Tm) can be calculated (Fig. 7).
1. Prepare 160 µL of GS-GN(Y195F) protein at 1.25 µM (working stock), by diluting with assay buffer.
2. Prepare G6P to 125 mM by adding 4 µL of the 1 M stock to 28 µL assay buffer.
3. Prepare your reaction plate: to a 96-well plate add 24 µL of protein at 1.25 µM working stock into six wells in the same row (A1 – A6) (Fig. 8). Add 24 µL of assay buffer to three more wells in the same row (A7 – A9). To the row below, add 6 µL of assay buffer into three wells (B1 – B3). Into the next three wells, add 6 µL of 125 mM G6P (B4 – B6). Into the next three wells, add 6 µL of assay buffer (B7 – B9).
4. Using a multichannel pipette, add 3 µL of B1 – B9 to A1 – A9.
5. Incubate the plate at room temperature for 30 minutes.
6. Add 5 µL of 25X SYPRO™ orange to the reaction plate (C1 – C9).
7. Use a multichannel pipette to transfer 18 µL of the GS-GN(Y195F) +/- G6P mixes (A1 – A9) into the final 96-well plate (A1* – A9*) (Fig. 8).
8. Use a multichannel pipette to add 2 µL of 25X SYPRO™ orange from the reaction plate to the final plate to all wells containing sample (C1 – C9 to A1* – A9*). This makes final concentrations of 1 µM GS-GN(Y195F), 12.5 mM G6P and 2.5X SYPRO™ orange.
9. Centrifuge plate at 800 g for 1 minute at room temperature.
10. Apply an adhesive sealing film on the top of the plate and ensure it is tightly secured.
11. Start a new programme on the Applied Biosystems QuantStudio 3 Real-Time PCR system. If access to QuantStudio 3 instrument and software are limited, other RT-PCR instruments can be used by adapting the protocol (see Note 11).
12. The program should first contain a PCR step to decrease the temperature of the machine to 10°C. Then run a melt curve stage using a gradient of 0.018°C/s from 20°C to 95°C, collecting data points using the filters x2(520 ± 10) and m2(558 ± 11). Select the wells to be recorded and specify these as targets. Ensure each target has the reporter set as ROX and the quencher set as none.
13. Start the method, and once the machine has reached 10°C and holding, pause the program and place your plate in the machine. Ensure your plate is inserted in the correct orientation, matching the A1 plate well to where A1 is specified in the machine drawer.
14. Continue the program.
15. Once the program has finished, transfer the results file to a USB pen.
16. Import the results file from the USB pen into the Protein Thermal Shift™ software.
17. Complete the plate template to specify what sample was in each well.
A1*-A3*= GS-GN(Y195F) – G6P
A4*-A6*= GS-GN(Y195F) + G6P
A7*-A9*= no protein control
18. Click analyse to generate raw, derivative and Boltzmann plots and Tm values and analysis (Fig. 9). You should see a curve similar to the curve shown in Fig. 7. Check that the no protein control is flat, and has a low absorbance reading at all temperatures.
19. You can export the file as .txt or .csv to perform plot in your chosen software (e.g. Excel or GraphPad Prism).
20. Repeat this experiment three times. Plot the mean ± SEM of three independent experiments completed in technical triplicates.
3.5 Quality control and concentration check of purified protein
1. Prepare bovine serum albumin (BSA) standard (solution) ready for loading at 0.01, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5 and 5.0 µg.
2. Dilute the purified protein to different desired concentrations within the BSA standard range.
3. Add 4X Laemmli buffer freshly supplemented with 100 mM DTT (to final concentration of 1X) to the BSA standards and purified protein and heat at 95°C for 5 minutes.
4. Leave the samples/tubes for a few minutes at room temperature and briefly spin down the condensates at 1000 g for 1 min.
5. Run the purified protein and BSA samples along with a protein standard marker on a 10% SDS-PAGE gel (see Note 10 and Note 12).
6. Stain the gel with the 0.25% Coomassie Blue R-250 dye solution for 2-4 hours or until the gel is uniformly stained (see Note 13).
7. Destain the gel overnight or until the background is clear in destaining solution (see Note 14).
8. Image/scan the gels and quantify the bands. Calculate and confirm the concentration of the samples from the BSA standard curve (Fig. 10).
3.6 Glycogen Synthase activity assay
GS glycosyltransferase activity is assessed by measuring the incorporation of 14C-glucose (from UDP-[U-14C]glucose) into glycogen as shown in the formula below.
1. Dilute 1 µg of purified GS-GN in ice-cold lysis buffer (with additives) to a total volume of 100 µL i.e. 10 µg/mL). For each reaction, you will need20 µL of the protein. (See Note 15 and Note 16). To determine specific activity, an extra 3 reactions are required – see below.
2. Prepare reaction buffer (For each reaction, you will need80 µL of reaction buffer).
3. For each sample dispense 80 µL reaction buffer with (+) or without (–) of G6P in duplicate (4 tubes in total). Pre-warm for 2-3 minutes at 30°C in an Eppendorf Thermomixer or water bath.
4. Start the assay by adding 20 µL diluted protein samples to each tube and vortex gently to mix. Include a negative control (lysis buffer without GS) (see Note 17). This is the assay mix.
5. Incubate the samples for 20-30 minutes at 30°C in an Eppendorf thermomixer or similar with gentle shaking at 300 rpm (see Note 18).
6. Stop the reaction by spotting 90 µL of the assay mix onto 2.5 cm x 2.5 cm squares of Whatmann 3MM paper. Soak the paper immediately into a beaker containing 8-10 mL of cold 66% (v/v) ethanol per paper (see Note 19).
7. Leave for ~20 minutes after the addition of the last paper.
8. Wash the filter papers at room temperature with 66% (v/v) ethanol (8-10 mL per paper) three times for 10 minutes per wash (see Note 20).
9. Rinse briefly with acetone and air-dry the filter papers.
10. To determine specific activity, spot 20 µL of undiluted reaction buffer onto filter papers in triplicate and air-dry without washing.
11. Transfer the filter papers to scintillation vials and add 4 mL scintillation fluid.
12. Use a scintillation counter to measure incorporated 14C (Fig. 11a).
13. Calculate specific activity (DPM.nmol-1)
14. Calculate activity (nmol.min-1.mg-1) (Fig. 11b) (see Note 21).
3.7 Notes
1. Check if HEPES is the free acid or the sodium salt and adjust the calculation accordingly if the molecular weight is different.
2. Check EDTA being used as it exists as multiple salts/hydrates and adjust the calculation accordingly if the molecular weight is different.
3. Check the pH of the assay buffer intermittently and check for visible salt precipitates, when storing the buffer for more than 6 months.
4. Determine optimal response to G6P by measuring enzyme activity in response to different concentrations of G6P. Using two different concentrations of the protein, determine GS activity in response to a range of G6P concentrations (0, 0.01, 0.03, 0.1, 0.25, 0.50, 0.75, 1, 3, 6, 12 mM). Refer to Fig. 11b.
5. Activate sodium orthovanadate by dissolving 7.36 g in 200 mL H2O and titrate the pH to 10 with 1 M NaOH or HCl until the solution turns yellow. Boil the solution in a microwave until it turns colourless and then cool to room temperature. Readjust the pH to 10 and repeat the steps from above until the solution remains colourless and the pH stabilises at 10. Aliquot and store at -20°C.
6. Make fresh DTT before the experiment. Alternatively, it can also be stored as small aliquots at -20°C for long term storage. Avoid repeated freeze thaw cycles.
7. Molecular weight of b-glycerophosphate disodium varies depending on its hydrate form. Check molecular weight of stock and adjust calculation accordingly.
8. For the transformation using chemically competent DH10Bac cells, we recommend using the following protocol: https://assets.fishersci.com/TFS-Assets/LSG/manuals/10361012.pdf
9. Multiple antibiotic resistances are used in the MultiBac system: ampicillin and gentamycin are used to screen for the presence of the gene of interest following integration of the pFastBac HTb or pFL plasmid into the modified baculovirus genome expressed by the DH10MultiBac cells. The modified baculovirus genome contains kanamycin and tetracycline resistance genes. Integration of the gene(s) of interest into the baculovirus genome via Tn7 transposition also disrupts lacZ expression, allowing blue/white screening Therefore, using all four antibiotics combined with blue/white screening will select for cells expressing the baculovirus genome into which the gene(s) of interest have been integrated.
10. For a typical SDS-PAGE protocol, we recommend using the following protocol: https://www.sigmaaldrich.com/GB/en/technical-documents/protocol/protein-biology/gel-electrophoresis/sds-page
11. Plate the reaction mixtures in a suitable 96-well plate for your RT-PCR machine and seal using clear adhesive. Design the program to pre-incubate the machine at 10°C, then to run a melt curve from 20°C to 95°C in 1°C increments. Set the program to collecting data points using the appropriate filters, and set the reporter as ROX and the quencher as none. Analyse the results file in your chosen software (e.g. Excel or GraphPad Prism) by plotting the fluorescence against temperature.
12. For better resolution of bands in the range of 35-40 kDa, let the 25 kDa band of the molecular weight ladder run just off the bottom of the gel.
13. Before staining, gels can be prefixed in 50% methanol, 10% acetic acid, 40% H2O for 30 minutes to overnight.
14. Gels can be stored for longer periods in 7% acetic acid.
15. Adjust volume according to the total number of assay conditions.
16. To determine enzyme linearity, determine enzyme activity using different concentrations of protein.
17. Allow 15-20 seconds interval between additions of samples.
18. The incubation time can be adjusted according to the experimental conditions and requirements.
19. Stir the liquid continuously by using a Teflon-covered stirring bar. Keep the stirring bar from physical contact with the samples by using a stainless-steelscreen inserted inside the beaker. Alternatively, place the beaker with the papers on a shaker with gentle shaking.
20. Include two blank filter papers as controls for washing efficiency.
21. While calculating the GS activity, a factor of 1.11 is multiplied to the corrected radioactivity counts (sample DPM - blank DPM) since the counts are measured in 90 µl of the 100 µl reaction spotted on the filter paper.