GSH/GSSG Assay

GSH/GSSG Assay

Solutions

  • Stock Buffers (A and B)
    • Prepare in water. Store at 4°C for ~6 months. May form a precipitate, just warm to 37°C when ready to use to dissolve this.
    • 27.6g/L – Sodium Phosphate (Monobasic) = A
    • 28.4g/L – Sodium Phosphate (Dibasic) = B
  • Working Buffer (AxB)
    • Phosphate Buffer w/0.005M EDTA
    • Make fresh each time (takes some time to dissolve)
    • 16ml "A" + 84ml "B" + 0.336g EDTA (Na2EDTA)
    • pH7.5
  • 6% SSA
    • (5-Sulfasalicylic acid) - 20mM in Working Buffer
    • Make fresh each time
    • 6gSSA/100ml Working Buffer) = 6%SSA
    • Re-suspend samples in 6%SSA, 1part SSA to 1part PBS, so final M=3%SSA. Dilute samples in 3%SSA to keep concentration.
  • DTNB
    • 0.6mg/ml, or 0.0006g/ml, in Working Buffer
    • Can make a 10x stock (6mg/ml, or 0.006g/ml) and store at -20°C, ~6months). For assay, dilute 10x in Working buffer
    • Need 100μl/rxn
  • NADPH
    • 1.667 mg/ml = 0.00167g.ml, in Working Buffer
    • Make fresh each time
    • Need 100μl/rxn
  • Glutathione Reductase (GR)
    • 100μg/ml= 0.0001g/ml in Working Buffer
    • 256U/ml stock = M1
    • Need 1.3U/rxn, or 1.3U/100μl = 13.3U/ml= M2
    • Need 100μl/rxn of this M2
  • GSH Stock
    • MW=307.3
    • 100mM , or 0.0307g/ml - In Working Buffer. Store Frozen
  • GSSG Stock
    • MW=612.6
    • 100mM, or 0.0613g/ml - In Working Buffer. Store Frozen

Standards
Prepare standards from stock by doing serial dilutions in 3%SSA Buffer

GSG GSSG
1 μM 0.25 μM
5 μM 0.5 μM
15 μM 2.5 μM
25 μM 7.5 μM
50 μM 15 μM

V1 = (M2V2)/M1
Diluent = V2 - V1

GSH - Stock 100mM = 100,000μM

V1 100μl 100μl 100μl
V2-V1 = Buffer + 900μl + 900μl + 1900μl
V2 1ml 1ml 2ml

V1 750μl 300μl 300μl
V2-V1 = Buffer + 750μl + 1200μl + 1200μl
V2 1.5ml 1.5ml 1.5ml

V1 300μl
V2-V1 = Buffer + 700μl
V2 1ml

GSSG - Stock 100mM = 100,000μM

V1 100μl 100μl 100μl
V2-V1 = Buffer + 900μl + 900μl + 1900μl
V2 1ml 1ml 1ml

V1 900μl 750μl 750μl
V2-V1 = Buffer + 600μl + 750μl + 750μl
V2 1.5ml 1.5ml 1.5ml

V1 750μl 500μl 300μl
V2-V1 = Buffer + 750μl + 1000μl + 1200μl
V2 1.5ml 1.5ml 1.5ml

V1 750μl 750μl
V2-V1 = Buffer + 750μl + 750μl
V2 1.5ml 1.5ml

Dilute Samples
If sample is not at least 180μl, you need to bring up the volume of sample to 180μl in 3% SSA Buffer before starting the assay. 100μl of sample will be used for GSSG assay and the rest for GSH. Make sure you note the Dilution Factor, as this will be used later to calculate the correct concentration of each.

Sample V1 V2 DF = V2/V1 V2-V1
1 180μl
2 180μl
3 180μl
4 180μl
5 180μl
6 180μl

GSSG Assay

  • Cell concentration = 1x107cells/ml = 10x106cells/ml = 1x106cells/100μ l rxn
  • After you have 180μl of each sample, aliquot 100μl for GSSG Assay. Keep all on ice!
  • Add the following to each of these Aliquots, GSSG Standards, Blank (100μl 6%SSA):
    • 100μl Sample (or Standard, or Blank)
    • 2μl 2-vp
    • 6μl Triethanolamine
    • 108μl total
  • Add this in hood. Use cut tips for Triethanolamine, since it is really viscous: take 6μl, clean tip, add to tube, and vortex.
  • Incubate for 1 hour on ice (can do GSH in meantime).

Assay

  • When assaying, each GSSG sample will be measured in duplicate. Three samples can be measured at a time, since you can fit 6 cuvets in spec. Note: You do not need to measure standards in duplicate.
  • Measure the standards first.
  • Set 6 bullet tubes on rack. Have 6 clean microcuvets ready near spec.
  • Add the following in the specified order to all six bullet tubes, except for the GR:
    1. Working Buffer - 673μl
    2. GSSG Sample/Std/Blank - 27μl
    3. DTNB - 100μl
    4. NADPH - 100μl
    5. GR - 100μl
  • Person1: When ready, add GR quickly to all six tubes, you don’t need to change tip.
  • Person2: Close each tube that is ready, vortex, decant into a clean cuvet, and place in spec. Start with slot farthest away from you, since this will be read first.
  • Note: Need to do all this quickly, since reaction starts as soon as you add the GR; however, make sure you do it carefully or you’ll mess up the entire run.

Measure

  • "Kinetics": A-412nm, at 25°C, 6 minutes. (Andre Nel's lab, can get program to set up on another spec)

Blank

  • Do this first. Use 27μl of incubated blank just like all other samples. Run first on spec, by itself, for 6 minutes.
  • First measure blank, then measure standards, and then samples (in duplicate).

Record Data

  • At the end of the run, record the dA for all that was measured. Later you will plot dA vs Concentration for standards to create a Standard Curve. The Std Curve will be used to calculate the Concentration of samples by plugging in dA into the equation of the curve.

GSG Assay

  • Follow similar procedure as for GSSG Assay
  • When assaying, each GSH sample will be measured in duplicate. Three samples can be measured at a time, since you can fit 6 cuvets in spec.
  • Measure the blank first. Then measure the standards. Then measure samples. Note: You do not need to measure standards in duplicate.
  • Blank: Use 25μl SSA Buffer instead. Run blank cuvet first, by itself, for 6 minutes.
  • Set 6 bullet tubes on rack. Have 6 clean microcuvets ready near spec.
  • Add the following in the specified order to all six bullet tubes, except for the GR
    1. Working Buffer - 675μl
    2. GSH Sample/Std/Blank - 25μl
    3. DTNB - 100μl
    4. NADPH - 100μl
    5. GR - 100μl
  • Person1: When ready, add GR quickly to all six tubes, you don’t need to change tip.
  • Person2: Close each tube that is ready, vortex, decant into a clean cuvet, and place in spec. Start with slot farthest away from you, since this will be read first.

Measure

  • "Kinetics": A-412nm, at 25°C, 6 minutes.

Data

  • Record dA for all measured.
  • Note: Spec records dA every 30 sec for the duration of the 360sec run. The dA you record will be the last one recorded by the spec, at 360sec.