MBL Protocol
                          2023 Barbora Hrnčířová


                                        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 miliQ H[2]O (pH adjusted with 5M HCl)


                1,5M Tris-HCl pH8,8 – 18,171g Tris base/100ml miliQ H[2]O (pH adjusted with 5M HCl)


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


                10% APS (as peroxoaminosulphate) – 0,1g/1ml miliQ 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 miliQ 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














Preparation of gels
  * if you plan to make more gels in a short time period, you can prepare a larger amount of the
solutions (without APS and TEMED) and keep them in the fridge (max 1,5 months).


Resolving gel stock (12%; 5 ml/gel)


Component

                    1 gel

                           2 gels

                                  4 gels

30% A/B

                    2 ml

                           4 ml

                                  8 ml

1.5M Tris-HCl pH 8.8

                    1.25 ml

                           2.5 ml

                                  5 ml

10% SDS

                    50 μl

                           100 μl

                                  200 μl

miliQ H2O

                    1.68 ml

                           3.35 ml

                                  6.72 ml


Stacking gel (4%; 2.5 ml/gel)


Component

                    1 gel

                           2 gels

                                  4 gels

30% A/B

                    0.33 ml

                           0.66 ml

                                  1.32 ml

0.5M Tris-HCl pH 6.8

                    0.63 ml

                           1.26 ml

                                  2.52 ml

10% SDS

                    25 μl

                           50 μl

                                  100 μl

miliQ H2O

                    1.5 ml

                           3 ml

                                  6 ml


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 the 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, and 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 the gel tray and insert the teeth (chambers)

·         Let gel polymerize for 30 min

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




Prepping the samples and running the gel:

  * Measure protein concentration in samples. In the case of cell lysates/CFEs, each sample of 20
μl should contain 8 μg of protein; for purified proteins, 4 μg is enough.
  * 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 a 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 the gel at 125 V, constant V until the loading dye (dark blue) reaches the end of the gel
(ca 80 min)


Staining the gel:

  * Disassemble the running apparatus, lift the small glass piece, and cut off 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, and 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