SDS – PAGE protocol


    Mini-PROTEAN Cells (BioRad)

    30% Acrylamide/Bis Solution 37.5:1 (BioRad - #1610158)

    Quick Coomassie stain (Serva - 35081.01)

    TEMED



Preparation of chemicals:


                0,5M Tris-HCl pH6,8 – 6,057g Tris base/100ml milliQ H[2]O


                1,5M Tris-HCl pH8,8 – 18,171g Tris base/100ml milliQ H[2]O



..pH adjusted with 5M HCl


                10% SDS – 10g/100ml milliQ H[2]O


                10% APS (as peroxoaminosulphate) – 0,1g/1ml milliQ H[2]O
1 month max! fridge


                Isobutanol, water saturated – 20 ml + 20 ml + shake (IsobutOH in upper phase)




Preparation of solutions:


                5x Running buffer           1g SDS

                                                                3g Tris base

                                                                14,4g Glycine

                                                                up to 200ml milliQ H[2]O


                5x Sample loading buffer             1,2 ml milliQ H[2]O

                                                                                0,5 ml of 0,5M
Tris-HCl ph6,8

                                                                                0,8 ml glycerol

                                                                                0,8 ml 10% SDS

                                                                                0,2 ml
β-mercaptoEtOH

                                                                                pinch of Bromphenol
blue


                Stacking gel stock (4%)                  1,98 ml 30%
A/B                                               1,5 month! fridge

                                                                                3,78 ml of 0,5M
Tris-HCl ph6,8

                                                                                150 µl 10% SDS

                                                                                9 ml milliQ H[2]O



                Resolving gel stock (12%)             6 ml 30% A/B
                    1,5 month! fridge

                                                                                3,75 ml of 1,5M
Tris-HCl ph8,8

                                                                                150 µl 10% SDS

                                                                                5,03 ml milliQ
H[2]O






Pouring the gel:


●        Set up the gel tray(s)

●        Pour 5ml of Resolving gel stock (per gel) into the 12% AB 15ml falcon tube

●        Add 50 µl of 10% APS and 8 µl of TEMED quickly and mix well

●        Immediately fill gel tray up to 1 cm under the teeth (chambers)

●        Carefully overlay the gel with 300 µl isobutanol using a syringe

●        Let gel polymerize for 1 hour

●        Absorb isobutanol using absorbent paper, rinse with dH[2]O, dry with absorbent paper again

●        Pour 2,5ml of Stacking gel stock (per gel) into the 4% AB 15ml falcon tube

●        Add 25 µl of 10% APS and 4 µl of TEMED quickly and mix well

●        Immediately fill gel tray and insert the teeth (chambers)

●        Let gel polymerize for 30 min

●        Remove the teeth, transfer the gel into the running apparatus, fill with 1x Running buffer


Prepping the samples and running the gel:


●        Mix your sample in Eppendorf tube with 5x Sample loading buffer (final conc. 1x) -
usually, a sample is mixed with water to give 20 ul and then 5 ul of loading buffer is added (12.5
ul is eventually loaded in single well of the gel)

●        Boil samples at 95°C for 5 min and centrifuge them briefly

●        Load samples and 5 µl of protein marker (keep on ice!)

●        Run gel at 25mA until the loading dye (dark blue) reaches the end of the gel


Staining the gel:


●        Disassemble the running apparatus, lift the small glass piece and cut of the separating
gel

●        Carefully push the gel into a small container with dH[2]O

●        Wash 10 min

●        Discard dH[2]O, add around 40 ml Quick Coomassie stain, stain for 1 hour +

●        Discard the stain (into a 50ml falcon tube for reuse)

●        Rinse the gel with dH[2]O a few times

●        Leave to de-stain in dH[2]O ON