I've been trying to perform a reduced GSH assay on some cell lysates and no colour is being produced upon the addition of DTNB (Ellmans). I have been adding 10uL of cell lysates to each well and adding 160uL of a solution consisting of pH7.0, 0.1M PBS buffer and DTNB. This protocol has been outlined on the Sigma-Aldrich site https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/cs0260bul.pdf .
I have been omitting the reductase and NADPH step as I only want reduced GSH for the meantime.
I put a few mg's of L-glutathione reduced (97%) into a small 1.5 eppendorf and pipetted ~ 40uL of DTNB, which resulted in a deep orange/red colour. I imagine this is a very concentrated version of what I'm after, but as soon as I added 50uL of 5% 5-Sulfosalicylic Acid (as mentioned throughout the protocol) I lose all colouration, same with the buffer.
Kind of at a wits end on this one, any help on this is greatly appreciated.
Edit: I'm not using a kit, my lab had the majority of the required chemicals and I have been adapting the protocol based of several protocols such as sigma's and cayman chemical's https://www.caymanchem.com/pdfs/703002.pdf .
Edit 2: all stock preps were made in the last month. I have yet to use any enzymes.
Dear Craig,
Determination of GSH is simple and easy:
1) At first add 1 mL TCA to 1 mL cell lysate or tissue homogenate.
2) Mix well and centrifuge 10 min at 5000 rpm and separate the supernatant deproteinization).
3) Add 1 mL DTNB to 1 mL of the supernatant, mix well, incubate 5 min at RT and read the absorbance at 412 nm (405-420 nm).
Note:
1) TCA is 10%
2) DTNB is 30mM (prepared in Citrate 0.1 preferably)
3) Use GSH 1 mM as standard
In my experiment OD of standard was around 0.570 and the samples around 0.280
This papers are very useful:
International Journal of Diabetes Research 2013, 2(3): 39-44
Reduced glutathione (GSH)was determined by the method of Ellman[19]. Briefly, 1 mL tissue homogenate was treated with 1 mL of 5 trichloroacetic acid (% 10) (Sigma, St. Louis, MO), the mixtures were centrifuged at 5000 rpm and the supernatant was taken. After deproteinization, the supernatant was allowed to react with 1 mL of Ellman’sreagent (30 mM 5, 5’-dithiobisnitro benzoic acid in 100 mL of 0.1% sodium citrate). The absorbance of the yellow product was read at 412 nm in spectrophotometer. Pure GSH was used as standard for establishing the calibration curve
Research Journal of Biological Sciences, 2010, 5(9), 615-624
Determination of reduced glutathione activity:
Reduced glutathione level was determined according the method
of Ellman (1959). The tissue homogenates (720 ll) was double
diluted and trichloroacetic acid (5%) was added to it to precipitate
the protein content of the tissue homogenates. After centrifugation
(at 11,112g, 5 min) the supernatant was taken, 5,5-dithiobis (2-
nitrobenzoic acid solution (Ellman’s reagent) was added to it and
the absorbance was measured at 417 nm. Reduced glutathione level
of homogenate was calculated using standard curve. A standard
curve was drawn using different known levels of reduced glutathione
solution
Another ref:
Principle:
Glutathione reacts with dithio bis nitrobenzoic acid (DTNB) to give a compound
that absorbs maximally at 412 nm.
Reagents
1. Phosphate Buffer: 0.2 M pH 8.0
2. DTNB: 0.6 mM DTNB in 0.2 M phosphate buffer, pH 8.0.
3. TCA: 5 %
4. Glutathione standard: 20 mg in 100 ml distilled water.
Procedure:
The proteins were precipitated with 5% TCA. The solution was mixed well and
centrifuged. To 0.5 ml of supernatant, 2 ml of phosphate buffer was added followed by 0.5 ml of DTNB reagent. The yellow color developed was read at 412 nm in a
spectrophotometer against a blank containing 5 % TCA instead of the sample. A series of standards were treated in a similar manner. The amount of glutathione in the hemolysate was expressed as μg of GSH /mg
Hb.
Best
mehdi
Wow, thank you very much for that very in-depth answer Mehdi. I will adjust my protocol accordingly, again thank you.
Total GSH was determined in the homogenates
spectrophotometrically at 412 nm, using yeast-
GR, 5,50-dithio-bis-(2-nitrobenzoic acid) (DTNB) and
NADPH (Anderson 1985).
This assay is very easy and simple. To check if the method is working use a serial dilution of standard GSH as control. If assay is working properly you will see yellow color product. Protein precipitation in the lysate is important by TCA. The Ellman;s reagent should be stored in a dark bottle.
Good luck
Dear Craig,
obviously both TCA and sulfosalicylic acid will change pH in your sample and you have to adjust pH around 7.0 prior your assay. This is not always written in protocols and I am more than sure it is making problem in your assay.
So, add neutralization agent after getting supernatant. The best neutralization you will achieve with salts originated from strong bases and weak acids (apart of neutralization they will maintain pH of your sample on the stable level working as a buffer), for example KHCO3 is good. Bases like KOH or NAOH are also ok.
Hope this will help.
Hai, this is not an answer. Just a question.
I just want to find out what lysis buffer to use to prepare cell lysate for determination of GSH. I was told that we should not use RIPA buffer. Thank you
I used the freeze thaw method to lyse my cells. Stored in -80 for an hour followed by ~20 minutes in a 37C heating block, for 5 cycles. If you have good LN2 facilities and appropriate equipment etc., I would recommend that over the -80 freezer. I had a 30% yield increase from using LN2 compared to the -80 freezer.
Though I only used the freeze thaw method due to the larger application of use compared to a lysing solution.
Hope this helps.
Its a question in relation to the above answer.
We are not using GR. So, Do the above method will quantify the total GSH concentartion?
With regards
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