SDS-PAGE

4X Resolving Buffer (1.5 M Tris-HCl, pH 8.8)

• Add 90.75 g to 400mL DI water
• Titrate the solution with ~18% HCl to pH 8.8
• Add water to a final volume of 500mL
• Store at 4°C

4X Stacking Buffer (0.5 M Tris-HCl, pH 6.8)

• Add 30.25 g to 400mL DI water
• Titrate the solution with ~18% HCl to pH 6.8
• Add water to a final volume of 500mL DI water
• Store at 4°C

10X Running Buffer

• Weigh 30 g Tris .
• Weigh 144 g .
• Weigh 10 g SDS.
• Dissolve in 1000mL water.
• pH should read 8.3. No pH adjustments are needed.
• Dilute to 1X before use.
• Store at room temperature.

10% Ammonium Persulfate (w/v)

• Add 1g of to 10mL of DI water
• Store at 4°C

10% SDS (w/v)

• Add 10g of to 100mL DI water
• Store at room temperature

3X Laemmli Buffer

• 2.4 mL 1 M Tris pH 6.8
• 3 mL 20% SDS
• 3 mL glycerol
• 1.6 mL beta mercaptoethanol
• a drop of bromophoenol blue

In an Eppendorf tube combine

• 0.5mL 30% Acrylamide: Bisacrylamide (29:1)
• 0.5mL DI water
• 10µL APS
• 1µLTEMED Pipette 200µL down the right side, and 200µL down the left side

Allow to solidify for 0h 5m 0s

Resolving Layer

This following recipe makes a 12% SDS-PAGE. For optimal resolution of large proteins (25-200 kDa), you should use smaller concentration of acrylamide (8%), and for resolution of small proteins (4-70 kDa), you should use higher percentage of acrylamide (12-15%). You can calculate a recipe for a different gel percentage using https://www.cytographica.com/lab/acryl2.html

Combine

• 1.645mL DI water
• 1.645mL 30% Acrylamide: Bisacrylamide (29:1)
• 1.25mL 4X Resolving Buffer (1.5 M Tris, pH 8.8)
• 50µL10% SDS
• 50µL 10% APS
• 5µL 4X Resol Buffer (1.5 M Tris, pH 8.8) TEMED must be used in a fume hood

Add APS just before casting as the gel begins to polymerize immediately after addition of APS.

Pour into the mold, leaving ~2 cm below where the bottom of the comb will be* Cover with a layer of isopropyl alcohol (IPA)

• Wait 0h 20m 0s for gel to solidify

Stacking Layer

The stacking layer helps all the proteins get lined up so all proteins enter the resolving layer at the same time

Dump out any excess IPA

Combine

• 2.6mL DI water
• 1mL30% Acrylamide: Bisacrylamide (29:1)
• 1.25mL Stacking Buffer (0.5 M Tris, pH 6.8)
• 50µL 10% SDS
• 50µL 10% APS
• 5µLTEMED TEMED must be used in the fume hood

Add APS just before casting as the gel begins to polymerize immediately after addition of APS.

Pour into the mold* Place the comb

• Wait 0h 15m 0s for gel to solidify
• Move to water storage

Preparation of Whole Cell Samples for SDS-PAGE analysis

Collect whole cell samples before and after induction, and normalize to the cell mass. This allows you to compare the protein expression before and after inducing cells.

Heat the waterbath or heatblock to 95°C* Read the optical density at 600 nm (OD600) of the cell culture using a photometer.

Note: the OD600 reading should be within the linear range of the photometer. If the reading appear to be lower than 0.1 or higher than 0.9 concentrate or dilute the cell sample accordingly in order to ensure the OD reading to fall within the 0.1- 0.9 range. If you dilute or concentrate the sample remember to calculate back the initial concentration (e.g. if you diluted 5 times the initial concentration is y*5, where y is the reading you have obtained from the diluted concentration)

Calculate the amount of culture to harvest to have a cell pellet equivalent to 1mL of OD600 =1.

         _Calculate the amount of biomass using the formula_ 



                    C<sub>i</sub>* V<sub>i</sub> = C<sub>f</sub>* V<sub>f</sub>



         _where C stands for "concentration", V stands for "volume", i stands for "initial" and f stands for "final". Therefore:_ 



                    y OD600 * x mL = 1 OD600* 1 mL 

         _where y is the OD600 reading and x is the volume to be calculated. Therefore_ 



                    x mL = ( 1 * 1 )/y

• Transfer the amount of culture as calculated into an appropriate tube and centrifuge it at 7,000 rpm for 0h 10m 0s in tabletop centrifuge
• Discard the supernatant

• Transfer 150µL of 1X Laemmli Sample Buffer in the tube and resuspend the pellet by pipetting.
• Transfer the tube to the water bath/heatblock, inserting it into the floaters and incubate the tube at 95°C for 0h 5m 0s
• Transfer the tube on ice for 0h 1m 0s
• Transfer the tube in a bench-top centrifuge and centrifuge at room temperature at max speed (e.g. 13,000 xg) for 0h 5m 0s
• Transfer the supernatant to a final 1.5 microcentrifuge tube. Note: the pellet won't be visible. Remove the supernatant without touching the bottom of the tube with the pipette tips to avoid carry-over of membranes. This step is necessary to remove membranes and debris that will affect a good quality run of the samples on the SDS-PAGE gel.
• Load 10µLof the supernatant on the SDS-PAGE gel.
• Store the remaining sample at -20°C.

Purified Protein

• Add 2.5µLof 3X Laemmli Buffer to 7.5µLof sample
• Incubate 0h 5m 0s at 95°C.
• Load the sample onto the SDS-PAGE gel.

Place in gel running box and cover with running buffer* If running only one gel, make sure the other side has a dummy gel cassette inserted.

• Fill the space between the two gels with 1X running buffer.
• Add 7.5µL ladder with dye to a well. Add 7.5 - 10 uL samples to wells.
• Run at 80V until the loading dye reaches the resolving/stacking layer interface.
• Run at 180V until the loading dye reaches the end of the gel.
• Remove gel from casing. Place in petri dish and cover with Coomassie blue dye. Allow to stain overnight.