# data[].attributes.body
http://www.assay-protocol.com/uploads/Clean_Lilaic/SDS%20PAGE.pdf
Use this,

# data[].attributes.body
The standards and unknown protein samples are treated in a boiling water bath for 5 min in electrophoresis buffer containing 5-10% 2-mercaptoethanol and 1% SDS.  Filter, or microfuge, out any precipitated protein.  An equal volume of 40% sucrose is then added to increase the sample density.  The sucrose solution also should contain 0.02% brom(o)phenol blue as a tracking dye.

# data[].attributes.body
-add 4.856g of Tris
-add 40mL of DI water

# data[].attributes.body
-add 6.04g of Tris
-add 50mL of DI water

# data[].attributes.body
-Dissolve 121.14 g Tris (American Bioanalytical #AB14042) in 800 ml dH₂O.

-Adjust pH to 7.0 with the appropriate volume of concentrated HCl. Bring final volume to 1 liter with deionized water

-Autoclave and store at room temperature.

# data[].attributes.body
15ml stock solution of western blot loading buffer. Dilute for use.

To prepare base solvent add 3ml 20% SDS

add 3.75mL 1M Tris buffer at pH 6.8 in a suitable container.

Add 9 mg bromphenol blue

2.4ml B-mercaptoethanol and mix well. (do this in the hood.) borrow from todd

Add 4.5mL 100%. glycerol to the solution, mix well.

Make up to a final volume of 15ml with dH20 and mix again thoroughly

Store at 4’C. Dilute to use.

# data[].attributes.body
How to dilute 40% Acrylamide/bis to 30%
1. Use C1V1=C2V2 Formula
2. 0.4x=0.3(14 *amount needed in mL)
3. x is the amount of Acrylamide needed
4.  the rest is D.I Water

How to dilute 20% SDS to 10% SDS
1. Half of the amount needed will be SDS and the rest will be D.I water

How to make APS
1. 0.1 g of Ammonium Persulfate
2. 1000 uL of D.I water

# data[].attributes.body
Resolving
30% Acrylamide/bis 0.5M 4 ml
h20 7.03 ml
Tris 3.75 ml
10% SDS  150 uL

Add exactly before pouring the gel:
10% APS 75 uL
TEMED 7.5 uL

Note: When you pour the gel, you need 7.5 mL of resolving.

Stacking
30% Acrylamide/bis 0.5M 1.3ml
h20 5.5ml
Tris 1ml (use pH 6.8)
10% SDS  80ul

Add exactly before pouring the gel:
10% APS 80ul
TEMED 8ul

Note:

# data[].attributes.body
Coomassie® R-250 Staining Protocol

1. Prepare the staining solution containing 0.1% Coomassie® R-250 in 40% ethanol, 10% acetic acid.

1. After electrophoresis, incubate 1 or 2 gels in a staining container containing 100 ml Coomassie® Blue R-250 staining solution. Caution: Use caution while performing the following steps using a microwave oven. Do not overheat the staining solutions.

1. Loosely cover the staining container and heat in a microwave oven at full power for 1 minute. To prevent hazardous, flammable vapors from forming, do not allow the solution to boil.

1. Remove the staining container from the microwave oven and gently shake the gel for 15 minutes at room temperature on an orbital shaker.

1. Decant the stain and rinse the gel once with deionized water.

1. Prepare a destain solution containing 10% ethanol and 7.5% acetic acid.

1. Place one or two stained gels in a staining container containing the 100 ml destain solution.

1. Loosely cover the staining container and heat in a microwave oven at full power for 1 minute.

1. Gently shake the gel at room temperature on an orbital shaker until the desired background is achieved.

# data[].attributes.body
Centrifuge the cell suspension at 2000G for 5-7 min at 4°C. The cells are collected at the bottom of the tube, discard the supernatant.

To the cell pellet, add ice-cold PBS and wash the cells by centrifuging at 2000G for 5-7 min at 4°C.

Add pop culture (10% of total volume ) to the cell pellet. Agitate the contents in microfuge tubes for 30 min at 27ºC.

Centrifuge the tubes at 16000G for 20 min at 4°C. Collect the supernatant in fresh tube and place on ice. Discard the pellet.

# data[].attributes.body
Load 10 ul of ladder
Load 10-25 of sample (25)
Run at 200V for 35 minutes

# data[].attributes.body
If not I can ask tomorrow / if its complicated

# data[].attributes.body
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006035.pdf

# data[].attributes.body
2 mL Tris (1 M, pH 6.8)

4.6 mL glycerol (50%)
1.6 mL SDS (10%)

0.4 mL bromophenol blue (0.5%)
(.02 g + 400 ul of H20)

0.4 mL β-mercaptoethanol

# data[].attributes.body
1. Centrifuge cells (~5 x 107) for 3 min at 15 ,000 x g and resuspend the pellet in an equal volume of 37°C PBS and centrifuge again. 2. Repeat two more times to remove all interfering material (extracellular proteases and growth media).

3. Add 200 μl of hot (95°C) SDS sample 10% solubilization buffer to the pellet and vortex thoroughly.

5. Cool the sample to 20°C and dilute with ~250 μl 2x SDS-PAGE sample buffer. Incubate for another 20 min at room temperature.

Centrifuge the sample solution at 20°C for 30 min at 14,000 x g and harvest the supernatant.

Perform the protein assay. The protein concentration should be ~5 μg/μl.

# data[].attributes.body
4 ml of H20
1.25 ml of 0.5M Tris pH 6.8
2ml of 50% glycerol (or sucrose)
2 ml of 10% SDS
.5 before use mercaptoethanol
.05g of bromophenol blue

# data[].attributes.body
10x
5 ml of 20% SDS
30g Tris
144 g of glycine
995 ml H20

1x
500 ul of 20% SDS or 1 ml of 10% SDS
3 g of Tris
14.4 g of glycine
999ml of H20 or to 1000

# data[].attributes.body
200 voltage for 30 min.
Well 1: ladder
Well 2: Supernatant cut+mhet
Well 3: Supernatant cut
Well 4: Supernatant PET
Well 5: Supernatant Ctrl
Well 6: Pellet cut+mhet
Well 7: Pellet Cut
Well 8: Pellet PET
Well 9: Pellet Ctrl

# data[].attributes.body
30% Acrylamide/bis : 12 ml
H2O: 21.09 mL
Tris: 11.25 mL
10% SDS: 450 uL

10% APS: 225 uL
TEMED: 22.5 uL

Approximately: 45 mL

Stacking
30% acrylamide/bis: 2.6 mL
H2O: 11 mL
Tris: 2 mL
10% SDS: 160 uL

10% APS: 160 uL
TEMED: 16 uL

# data[].attributes.body
.976 Control
.862 Cutinase & Mhetase
.873 PETase
.811 Cutinase

# data[].attributes.body
30% Acrylamide/bis 0.5M 1.3ml
h20 5.5ml
Tris 1ml (use pH 6.8)
10% SDS  80ul

10% APS 80ul
TEMED 8ul

# data[].attributes.body
If you want to make quick extracts from whole cells, I would suggest to take 1ml of culture at OD(600)=2.0 (or more or less depending on the density of your culture), spin down the cells and resuspend the pellet in 50 µl of 1xSDS loading buffer. Boil for 10 min, then spin at max. speed for 10 min. The sample will be viscous, but if you take 8 µl from the very top of the liquid, you should easily be able to pipet it.

I always use this protocol to look at the total lysate and I never have problems with it. If you stick to these values for the OD and for the volumes, then you should get a nice Coomassie-stain, and Western blot should also work perfectly...

# data[].attributes.body
30% Acrylamide/bis 0.5M 4 ml
h20 7.03 ml
Tris 3.75 ml( pH 8.8)
10% SDS  150 uL

10% APS 75 uL
TEMED 7.5 uL

Approximately 15ml
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdf

Resolving to 40% to 30%
3ul -Pa x 3 = 9mL
1ul -H20x3 = 3mL

Stacking 40% to 30%
.975uL Pa x3= 2.925
.325 uL H2o x 3 = .975uL

# data[].attributes.body
30% Acrylamide/bis 0.5M
(0.5 x X%)

Tris-HCl, pH 6.8 (Stacking)

1.5M Tris-HCl, pH 8.8 (resolving) 3.75 ml

10% SDS 150 μl

diH2O 7.28 ml
(11.03–(0.5 x X%))

TEMED 7.5 μl

10% APS 75 μl

Total volume 15ml

# data[].attributes.headline
SDS -PAGE (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis Notes and protocol (7/15)

# data[].attributes.subject
SDS -PAGE (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis Notes and protocol (7/15)

# data[].attributes.body
Mutant/Non-Mutant PETase
The Sequence Manipulation Suite: Protein Molecular Weight
Results for 873 residue sequence starting "ATGAATTTCC".
The protein weighs 72.69 kilodaltons

MHETase
The Sequence Manipulation Suite: Protein Molecular Weight
Results for 1904 residue sequence starting "ATGCAGACCA".
The protein weighs 157.34 kilodaltons

4-40 kDa | Up to 20%
12-45 kDa | 15%
10-70 kDa | 12.5%
15-100 kDa | 10%
50-200 kDa | 8%
>200 kDa | 4-6%

We are using an 8% gel

https://www.cytographica.com/lab/acryl2.html
Link to determine variable percentages for resolving gel