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[matthew.ngkw]

Hi everyone, i'm a biotechnology student currently on work experience internship.

My project is about extracting high molecular weight (HMW) glutenin subunit proteins from wheat grains for MALDI-TOF analysis

In short, i have adapted the method from the paper as shown in the link but I am not getting the quality and consistency of results as reported.
http://life.cnu.edu....70629100634.pdf

Some information on my project:
1) Extraction has to be done from half seeds of grain, about 20mg
2) HMW glutenins take up the smallest proportion of proteins
3) Glutenin subunits Mr ranges from 60 kDa to 95 kDa
4) Subunits form intermolecular disulfide bonds to form long polymers in native form and are thus highly hydrophobic
5) pI of glutenins are around 6.5 to 7.0 (i am not certain of this)

Summary of my method

1) Grind 20mg half seed into fine powder
2) Add 1ml 0.3M NaI - 7.5% propanol (this step is to wash off gliadins - monomeric proteins which are of greatest abundance)
3) centrifuge and discard supernatent
4) 300ul of 50% propanol, 0.08M Tris-HCl pH 8.0, 1% DTT, 1% SDS is added to the pellet. (DTT is added fresh. Used as a reductant to reduce disulfide bond to break up disulfide bonds to yield separate subunits and to improve solubility. SDS is added to improve extraction/solubility as well.)
5) centrifuge and supernatant is collected (pellet discarded)
6) HMW glutenin subunits are selectively precipitated by adjusting supernatant to 40% acetone (by adding 100% acetone)
7) Precipitation is carried out overnight at -20 degrees Celcius
8) Mixture is centrifuged (white pellet observed) and supernatant is discarded
9) steps 4-8 are repeated (second acetone precipitation supposedly remove contaminating pellet/salts? is this even necessary for MALDI-TOF analysis? because i think i am losing some proteins here)
10) Precipitate is air dried and then resuspended in 30% v/v Acetonitrile, 0.4% v/v TFA (with water as solvent)
11) Samples are mixed with Sinapinic acid matrix (10mg/ml of 50% acetonitrile, 50% water)in 1:10 ratio
12) mixture spotted via drip droplet method

I noticed that the pellet takes much longer than 1 hour (as reported in most papers)for final resuspension after the acetone precipitation even with agitation. Sometimes pellet does not fully redissolve. Will washing pellet with milli Q water after acetone precipitation help with this?

Generally, i am getting very poor signal strength. i occasional get some decent profiles but they are too inconsistent. Can anyone advise on this method? increasing starting sample quantity is not an option.

Thanks in advance. Any help is greatly appreciated!

Regards,
Matthew