Lab Resources

1. Cut PL+MG samples and place in 2 mL tubes
a. Cut to size of an eraser on a pencil if possible
b. Keep on dry ice until you cut
2. Add Lysis Buffer (400 uL) then homogenize
a. Lysis Buffer:
Trition 100 uL
1.5M NaCl 100 uL
PMSF 100 uL
Tris Base 20 uL
Protease Inhibitor 2 uL
Phophotase Inhibitor 2 uL
Water 676 uL
1000 uL
3. Centrifuge + Decant protein into .5 mL tubes
a. Aliquot 50 uL of protein per tube and freeze at -80˚C or leave on ice to use right away
4. Pull out plastic 96 well plate
5. Make master mix on right for each standard / protein sample
6. Add 5 uL of each standard / protein sample to wells (x3)
Standard 1 (A 1-3) 5 uL LysisStandard 2 (A 4-6) 10 uL Stock, 90 uL Lysis
Standard 3 (A 7-9) 25 uL Stock, 75 uL Lysis
Standard 4 (A 10-12) 50 uL Stock, 50 uL Lysis
Standard 5 (B 1-3) 75 uL Stock, 25 uL Lysis
Protein (B 4-?) 2 uL Protein, 18 uL Lysis
7. Record where you put each sample
8. Add 25 uL of working solution
a. Working Solution:
b. Reagent S (10 uL) Reagent A (1 mL)
9. Add reagent B (200 uL)
10. Incubate at room temp – 10 min
11. Use Spectrophotometer to create data
a. Wavelength for protein = 750 nm
12. Calculate data on spreadsheet

1. Prep samples with calculated values
a. Sample buffer
b. Water
c. uL of protein sample
2. Heat at 95˚C – 10 min
3. Running the gel
a. Assemble apparatus and fill with running buffer
b. Load samples into gel
c. Load ladders (Biorad, GE)
d. Run gel @ 100 V – 70 min
4. Transfer the gel
a. Assemble apparatus and fill with transfer buffer
b. Sandwich= Clear Side (down), Sponge, Filter, Membrane, Gel, Filter, Sponge, Black Side (up)
c. Remove bubbles and place cassette in apparatus
d. Add stir bar and ice pack to apparatus
e. Run gel @ 100 V – 65 min
5. Remove gel and cut membrane to size of western case
6. Block with Milk – 30 min
a. Milk = 2.5 g milk in 50 mL TBST
b. Rinse w/ TBST – 1x to remove milky residue
7. Add Primary Antibody in BSA – 60 min or overnight
a. BSA = 2.5 g BSA in 50 mL TBST
b. 4-5 mL per blot
c. Rinse w/ TBST – 3x3 min
8. Add Secondary Antibody in Milk – 60 min
a. Concentration 1:10,000 (5 mL per blot)
b. If Rabbit 1˚ = anti-donkey/anti-rabbit 2˚
c. If Mouse 1˚ = anti-donkey/anti-mouse 2˚
d. Rinse w/ TBST – 3x3 min
9. ECL – 5 min
a. 40 buffer : 1 substrate
b. 3-4 mL per blot
10. Cover in plastic + Dry ECL out + Put in Cassette
11. Dark Room Developing – 10 sec (may need multiple times)
12. Analyze blots with densitometry to create data
a. Expose and analyze dark to light comparisons
b. Rinse w/ TBST – 3x3 min
13. Stripping buffer – 10 min
a. Rinse w/ TBST – 3x3 min
14. Block w/ Milk in incubator chamber – 30 min
a. Rinse w/ TBST – 1x to remove milky residue
(Add next primary antibody + leave in 5^th floor fridge on rocker overnight)

1. Section tissue onto slides
a. Prepare sample in chamber – 30 min
b. Orient to view both PL + MG
2. Dewax (hospital slides only)
a. 2x clear rite – 5 min
b. 2x 100% etOH – 2 min
c. 1x 95% etOH – 1 min
d. 1x 70% etOH – 1 min
e. Rinse in DI H2O – 5 min
3. Rodent Decloaker – 20 min
a. Almost boiling in microwave (95˚C)
4. Cool Down – 10 min
a. Rinse in H₂O – 2x3 min
5. Use Pappen to circle tissue
6. Peroxidaze – 10 min
a. Rinse in TBS – 2x3 min
7. TBST – 10 min
a. 200 uL on each sample
8. Rodent Block – 15 min
a. Rinse in TBS – 2x3 min
9. Add Primary Antibody – 1 hour
a. 200 uL on each sample
b. Use universal neg. ctrl for ctrl slides
c. Rinse in TBS – 2x3 min
10. Add Secondary Antibody – 30 min
a. If Rabbit 1˚ = anti-donkey/anti-rabbit 2˚
b. If Mouse 1˚ = anti-donkey/anti-mouse 2˚
c. Use universal neg. ctrl for ctrl slides
d. Rinse w/ TBS – 2x3 min
11. DAB – 5 min
a. 1 mL substrate : 1 drop of Chromagen
b. Rinse w/ TBS – 2x3 min
12. Hematoxylin – 1 sec
a. Rinse in running H₂O – 5 min
13. Mount with cover slip and let dry
14. Take pictures on microscope

10% SDS-PAGE

Protein Gel:

We are using a discontinuous gel, so it is best to use a stacking gel in addition to the resolving gel
The protein will accumulate in the stacking gel before separating.

Resolving gel:

TOTAL 15ml

· 3.75 ml of 1.5M Tris.
· 3.75 ml of 40% acrylamide *
· 150 ml of 10% SDS
· 7.35 ml of H2O

Add later when ready to pour gel

· 50 ul of 10% APS
· 10 ul of TEMED **

Acrylamide is toxic and carcinogenic. Wear gloves, eye protection, and lab coat when using.
TEMED is toxic, Wear gloves, eye protection, and lab coat when using.

Add the gel solution to the disposable cassette using a 10ml pipette and slowly pour gel to the top line of the cassette (like 1mm from top). Fill the top of cassette with 50/50 H2O/EtOH solution. This solution will help to flatten the top of the gel. Polymerization will probably take 30 minutes.

Stacking Gel:

TOTAL 5ml

· 1.25ml 0.5M Tris
· 488ml of 4% acrylamide
· 50ml of 10% SDS
· 3.162ml of H2O
· 25ul of 10% APS
· 5ul of TEMED

Remove the 50/50 H2O/EtOH solution and pour your stacking gel on top of the set resolving gel using a 5ml pipette and place your comb. Let it polymerize for 30 minutes.

Materials needed:

· Tris base (Fisher BP152-500)
· 40% acrylamide solution (Fisher BP1408-1)
· SDS (Fisher BP166-100)
· APS (Fisher BP179-100)
· Temed (Fisher BP150-20)
· Ethanol (Fisher A407-1)
· HCl (Fisher A144-500)
· Disposable cassettes (Invitrogen NC 2010)
· Serological pipettes

Solutions:

50/50 EtOH

· 50 ml H20
· 50 ml EtOH

Store at room temperature

1.5M Tris pH 8.8

45.5g of Tris Base in 100ml H2O, adjust pH to 8.8 with 6N HCl, and bring to volume to 250ml with di H2O. Filter solution through 0.45uu filter.

13% SDS-PAGE

Protein Gel:

We are using a discontinuous gel, so we will use a stacking gel in addition to the resolving gel:

The protein will accumulate in the stacking gel before separating.

Resolving gel:

TOTAL 15ml
· 3.75 ml of 1.5M Tris.
· 4.875 ml of 40% acrylamide *
· 150 ml of 10% SDS
· 6.225 ml of H2O
Add later when ready to pour gel
· 50 ul of 10% APS
· 10 ul of TEMED **

Acrylamide is toxic and carcinogenic. Wear gloves, eye protection, and lab coat when using.
TEMED is toxic, Wear gloves, eye protection, and lab coat when using.

Add the gel solution to the disposable cassette using a 10ml pipette and slowly pour gel to the top line of the cassette (like 1mm from top). Fill the top of cassette with 50/50 H2O/EtOH solution. This solution will help to flatten the top of the gel. Polymerization will probably take 30 minutes.

· 50/50 H2O/ EtOH
· 50 ml of H2O
· 10 ml of EtOH

Stacking Gel:

TOTAL 5ml
· 1.25ml 0.5M Tris
· 488ml of 4% acrylamide
· 50ml of 10% SDS
· 3.162ml of H2O
· 25ul of 10% APS
· 5ul of TEMED

Remove the 50/50 H2O/EtOH solution and pour your stacking gel on top of the set resolving gel using a 5ml pipette and place your comb. Let it polymerize for 30 minutes.

Materials needed:

· Tris base (Fisher BP152-500)
· 40% acrylamide solution (Fisher BP1408-1)
· SDS (Fisher BP166-100)
· APS (Fisher BP179-100)
· Temed (Fisher BP150-20)
· Ethanol (Fisher A407-1)
· HCl (Fisher A144-500)
· Disposable cassettes (Invitrogen NC 2010)
· Serological pipettes

Solutions:

50/50 EtOH

· 50 ml H20
· 50 ml EtOH
· Store at room temperature

1.5M Tris pH 8.8

45.5g of Tris Base in 100ml H2O, adjust pH to 8.8 with 6N HCl, and bring to volume to 250ml with di H2O. Filter solution through 0.45uu filter.

* Wear gloves, eye protection, and lab coat while following this protocol.

This method is used to transcribe total RNA or mRNA to cDNA. You will need the following for each 20ul reaction:

· 10 pmol oligo d(T)15 = 1ul
· 10 ug RNA (in <5ul) = ____
· 5X AMV RT buffer = 4 ul
· 10mM dATP, dTTP, dCTP, dGTP = 1 ul each (4 ul total)
· Rnasin = 0.5 ul
· AMV-RT = 4 ul
· DEPC H20 to 20 ul = ____
· 20 ul

1. Prepare a master mix of all ingredients except RNA and DEPC H20 for as many reactions as you will have. Do this on ice.
2. Label 0.5ml tubes with sample data and add the appropriate amount of RNA and water.
3. Add 13.5 ul of master mix to each tube.
4. Incubate at 42°C for 2 hours.
5. Place samples at 95°C for 1-2 min., then chill on ice for at least 1 minute.
6. Freeze at -20°C if not used right away for PCR.

Materials needed:

1. Oligo d(T) 15 primer (Promega #C1101)
2. AMV reverse transcriptase and 5X buffer (Promega #M5101)
3. Rnasin (Promega #N2111)
4. dATP, dTTP, dCTP, dGTP (Invitrogen or Sigma – make sure it is 10mM)
5. DEPC water
6. micro centrifuge tubes
7. water bath at 42°C
8. heat block at 95°

Bacterial culture stocks:

Make a solution of 7% dimethylsulphoxide (DMSO) in LB broth.

Freeze cultures in -80˚C.

1. For this experiments we will use the following conditions for osmotic stress:

2. 700 mosmol in the medium. The medium has 300 mosmol/ kg of H2O so:
Stock = 500 mosmol/ml (14.6 % NaCL)

· (5 mosmol/ml) X = (.4 mosmol/ml) (50 ml)
· X=4 ml of stock + 46ml of medium (10 % FBS)
1,100 msomol
· (5 mosmol/ml) X = (.8 mosmol/ml) (50 ml)
· X=8 ml of stock + 42 ml of medium (10 % FBS)

3. 1,500 msosmol

· (5 mosmol/ml) X = (.8 mosmol/ml) (50 ml)
· X=10 ml of stock + 40 ml of medium (10 % FBS)

n=1
n=2

4. We will do an n=2 so is one 6 well plate for treatment. Treatment will be for 2, 4, 6, 8 and 24 hours.

Total of plates needed: 20 plates

Treatment plates:

· 3 for 2 hours
· 3 for 4 hours
· 3 for 6 hours
· 3 for 8 hours
15
3 for 24 hour

Control plates

· 1 for 2 hours
· 1 for 4 hours
· 1 for 6 hours
· 1 for 8 hours

5
1 for 24 hour

5. Cell will be plated at 200, 000 cells/well and let them grow for 48 hours before the experiment (around 70-80 % confluency).

6. On the day of the experiment media will be change and cells will be lysed with 50 ul of lysis buffer. Important to remember that the media need to be change in the controls plates also!!! Samples will be spun at 200 RPM for 5 min. Remove the supernatant and store the samples at -80°C.

7. Media should be saved and put on ice to measure osmolarity in the osmometer by Karen.

8. Protein lysates will be quantified and western should be run for:

a. NFAT5
b. AR
c. SMIT

* Wear gloves, eye protection, and lab coat when performing this procedure

1. 24- well microtitre plate assay: Add 50 μl of lysis buffer +100 μl of cleavage buffer.
2. Freeze/thaw cycle at -80ºC for 30 minutes and 10 minutes at 37ºC (water bath), repeat three times.
3. Add 34 μl of ONPG, incubate for 50 minutes at 37ºC.
4. Add 250 μl of stop buffer. Wait for ten minutes.
5. Transfer 150 μl of the supernantant from the 24-well plate to a 96-clear bottom plate.
6. Read plate at 420nm.
7. Calculate the amount of ONPG hydrolyzed = (OD₄₂₀)(3.84*10⁵nl)/(4500 nl/nmole-cm) (1cm).
8. To determine specific activity of beta-galactosidase (nmoles of ONPG hydrolyzed per minute per mg of protein):Specific activity = nmoles ONPG hydrolyzed/t/mg protein where t=the time of incubation in minutes at 37ºC and mg of protein is the amount of protein assayed.

Material/Supplies Needed:

1. Beta-galactosidase Assay (Invitrogen #K1455-01)
2. 24 well microtiter plate
3. -80˚C freezer
4. 37˚C water bath
5. 96 well microtiter plate
6. Plate reader with 420nm filter