User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2015/04/08

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Running SDS-PAGE

Dr Hartings' protocol can be found here.

April 8th

SDS Page Gel Electrophoresis

I. A 10x SDS-PAGE Running Buffer (30.3 g Tris, 144.1g Clycine, 10g SDS, water to 1L) was diluted by a factor of 10.

  • 100 mL of the 10x buffer was added to a 1000 mL volumetric flask

II. 20 μL of each of the samples prepared yesterday, in addition to a ladder/marker, was loaded into one of four wells in the following order:

  • The samples were heated for 5 min at 100C in the thermocycler pre-loading

Gel AO

Well Sample
1 Ladder
2 1 μM Proteinase K - Dilute
3 100 nM Proteinase K - Dilute
4 10 nM Proteinase K - Dilute
5 1 nM Proteinase K - Dilute
6 Blank
7 1 μM Proteinase K
8 100 nM Proteinase K
9 10 nM Proteinase K
10 1 nM Proteinase K


Gel AI

Well Sample
1 Ladder
2 Blank
3 1 μM Trypsin - Dilute
4 100 nM Trypsin - Dilute
5 10 nM Trypsin - Dilute
6 1 nM Trypsin - Dilute
7 Blank
8 1 μM Trypsin
9 100 nM Trypsin
10 10 nM Trypsin
11 1 nM Trypsin


Gel BI

Well Sample
1 Ladder
2 Blank
3 1 μM Chymotrypsin - Dilute
4 100 nM Chymotrypsin - Dilute
5 10 nM Chymotrypsin - Dilute
6 1 nM Chymotrypsin - Dilute
7 Blank
8 1 μM Chymotrypsin
9 100 nM Chymotrypsin
10 10 nM Chymotrypsin
11 1 nM Chymotrypsin


Gel BO

Well Sample
1 Ladder
2 Blank
3 1 μM Thermolysin - Dilute
4 100 nM Thermolysin - Dilute
5 10 nM Thermolysin - Dilute
6 1 nM Thermolysin - Dilute
7 Blank
8 1 μM Thermolysin
9 100 nM Thermolysin
10 10 nM Thermolysin
11 1 nM Thermolysin

III. 4 - 12 well pre-cast Mini Protean TGX gel was obtained and prepared.

  • The comb and tape was removed from each gel
  • The wells of each gel were rinsed with the prepared running buffer dilution

IV. The Bio-Rad Mini Protean system electrophoresis cell was assembled.

  • The inner and outer buffer chambers were filled with the diluted running buffer to the "4 gel" mark.

NOTE:The cell was initially prepared incorrectly, the "shorter wall" of the gel was supposed to face inside, not outside. The orientation of the gels was switched due to loading error and potentially contributed to the poor results.

V. The gel was run for approximately 40 min at 200 V, 0.05 A, and 10 W.

NOTE:Only the B side gel appeared to be running for the first approx. 20 minutes; adjustments were made but the A side gels ultimately only ran for about 20 min instead of the suggested 40.

VI. After 40 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 20 minutes

VII. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 20 minutes

VIII.Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) for 20 minutes and the results are pictured below:

NOTE:The fixing, staining, and de-staining processes were shortened because of time limitations. Additionally, the results demonstrate what appears to be primarily protein ladder/marker that made its way into adjacent wells, and nothing else.




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