TITLE: SDS-PAGE
AUTHORS: Anna Bird, Chiara Gandini

[DESCRIPTION]
SDS-PAGE gels are used to visualize proteins. This protocol describes how to prepare all the buffers required for casting and running SDS-PAGE gels, as well as how to prepare whole cell samples.

[STEPS]
SECTION: Buffers
1. 4X Resolving Buffer (1.5 M Tris-HCl, pH 8.8)
Add 90.75 g   to 400 mL  DI water
Titrate the solution with ~18% HCl to pH 8.8
Add water to a final volume of 500 mL 
Store at 4°C



SECTION: Buffers
2. 4X Stacking Buffer (0.5 M Tris-HCl, pH 6.8)
Add 30.25 g   to 400 mL  DI water
Titrate the solution with ~18% HCl to pH 6.8
Add water to a final volume of 500 mL  DI water
Store at 4°C
SECTION: Buffers
3. 10X Running Buffer
Weigh 30 g Tris  .
Weigh 144 g  .
Weigh 10 g    SDS.
Dissolve in 1000 mL  water.
pH should read 8.3. No pH adjustments are needed.
Dilute to 1X before use.
Store at room temperature.





SECTION: Buffers
4. 10% Ammonium Persulfate (w/v)
Add 1g of   to 10 mL of DI water
Store at 4°C


SECTION: Buffers
5. 10% SDS (w/v)
Add 10g of    to 100 mL DI water
Store at room temperature


SECTION: Buffers
6. 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
SECTION: Gel Casting
7. In an Eppendorf tube combine
0.5 mL 30% Acrylamide: Bisacrylamide (29:1)
0.5 mL DI water
10 µL APS
1 µLTEMED
Pipette 200 µL down the right side, and 200 µL down the left side
Allow to solidify for 5 min



SECTION: Gel Casting
8. 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
SECTION: Gel Casting
8.1. Combine
1.645 mL DI water
1.645 mL 30% Acrylamide: Bisacrylamide (29:1)
1.25 mL 4X Resolving Buffer (1.5 M Tris, pH 8.8)
50 µL10% SDS
50 µL 10% APS
5 µL TEMED
TEMED must be used in a fume hood
Add APS just before casting as the gel begins to polymerize immediately after addition of APS.





SECTION: Gel Casting
8.2. 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 20 min for gel to solidify

SECTION: Gel Casting
9. Stacking Layer

The stacking layer helps all the proteins get lined up so all proteins enter the resolving layer at the same time
SECTION: Gel Casting
9.1. Dump out any excess IPA

Combine
2.6 mL DI water
1 mL30% Acrylamide: Bisacrylamide (29:1)
1.25 mL Stacking Buffer (0.5 M Tris, pH 6.8)
50 µL 10% SDS
50 µL 10% APS
5 µLTEMED
TEMED must be used in the fume hood
Add APS just before casting as the gel begins to polymerize immediately after addition of APS.





SECTION: Gel Casting
9.2. Pour into the mold
Place the comb
Wait 15 min for gel to solidify
Move to water storage


SECTION: Sample Preparation
10. 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.
SECTION: Sample Preparation
10.1. 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)

SECTION: Sample Preparation
10.2. 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
                        Ci * Vi = Cf * Vf
            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 10 min in tabletop centrifuge
Discard the supernatant

SECTION: Sample Preparation
10.3. 
Transfer 150 µL 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 5 min
Transfer the tube on ice for 1 min
Transfer the tube in a bench-top centrifuge and centrifuge at room temperature at max speed (e.g. 13,000 xg) for 5 min
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 10 µLof the supernatant on the SDS-PAGE gel.
Store the remaining sample at -20 °C.

SECTION: Sample Preparation
11. Purified Protein
Add 2.5 µLof 3X Laemmli Buffer to 7.5 µLof sample
Incubate 5 min at 95 °C.
Load the sample onto the SDS-PAGE gel.
SECTION: Running the Gel
12. 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 7.5 µL 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.