App Moisture 105
App Moisture 105
App Moisture 105
Analytical Methods
Calculation
Moisture (%) = (W1-W2) x 100
W1
where:
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Method
Weighed 1 g of sample (mince or gel). Cut into small pieces and place in a
100 ml conical flask. Add 20 ml of solvent and homogenize for 1 min. Heat the
mixture in boiling water bath for 2 min. Stir at room temperature for 4 h. Centrifuge at
10,000xg for 30 min in a Sorvall-RC2 centrifuge. To 10 ml of the supernatant (soluble
fraction), add cold 50 % (w/v) TCA (2 ml). Kept the mixture at 4C for 18 h and
centrifuge at 10,000xg for 20 min.
Method
1. To 0.5 ml of sample, add 2.0 ml of the biuret reagent and mixed well.
2. Incubate the mixture at room temperature for 30 min.
3. Read the absorbance at 540 nm.
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water (l)
500
400
100
300
200
200
300
100
400
500
10
Method
1. Homogenize 10 g of muscle in 100 ml chilled (4C) 0.6 M KCl, pH 7.0
for 4 min.
2. Place the beaker containing the sample in ice. Each 20 sec of blending was
followed by a 20 sec rest interval to avoid overheating during extraction.
3. Centrifuge the extract at 5,000xg for 30 min at 4C.
4. Add three volumes of chilled distilled water to precipitate actomyosin.
5. Collect actomyosin by centrifuging at 5,000xg for 20 min at 4C.
6. Dissolve the pallet by stirring for 30 min at 4C in an equal volume of
chilled 0.6 M KCl, pH 7.
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1 % (w/v) SDS
TEMED (N,N,NN-tetramethylenediamine)
Sample buffer: Mix 30 ml of 10 % of SDS, 10 ml of glycerol, 5 ml of mercaptoethanol, 12.5 ml of 50 mM Tris-HCl, pH 6.8, and 10 mg
Bromophenol blue. Bring the volume to 100 ml with distilled water and
stored at -20C.
Method
Pouring the separating gel:
1. Assemble the minigel apparatus according to the manufacturers detailed
instructions. Make sure that the glass and other components are rigorously
clean and dry before assembly.
2. Mix the separating gel solution by adding, as defined in following Table.
3. Transfer the separating gel solution using a pasture pipette to the center of
sandwich is 2 cm from the top of the shorter glass plate.
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4. Cover the top of the gel with a layer of isobutyl alcohol by gently
squirting the isobutyl alcohol by gently squirting the isobutyl alcohol
against the edge of one of the spacers. Allow the resolving gel to
polymerize fully (usually 45 min).
Pouring the stacking gel:
1. Pour off completely the layer of isobutyl alcohol.
2. Prepare a 4 % stacking gel solution by adding as defined in table.
3. Transfer stacking gel solution to tickle into the center of the sandwich
along an edge of one of the spacers.
4. Insert a comb into the layer of stacking gel solution by placing one corner
of the gel and slowly lowering the other corner in. Allow the stacking gel
solution to polymerize 45 min at room temperature.
Reagents
30 % Acrylamide-bis
10 % running gel
3.333 ml
2.5 ml
10 % SDS
Distilled water
4 % stacking gel
0.665 ml
1.25 ml
100 l
50 l
4.012 ml
3 ml
50 l
25 l
5 l
3 l
2 % Ammonium persulfate
TEMED
Sample preparation:
1. Weigh 3 g of sample and homogenize with 5 % (w/v) SDS in a final
volume of 30 ml.
2. Incubate the mixture at 85C for 1 h.
3. Centrifuge at 3,500xg for 5 min at ambient temperature and collect
supernatant.
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of absorbent paper or paper towels. Carefully slide the spacers out from
the edge
the sandwich along its entire length.
3. Insert a spatula between the glass plates at one corner where the spacer
was, and gently pry the two plates apart.
4. Remove the gel from the lower plate. Place the plate with the gel attached
into the shallow dish of fixing agent of dye and swishing the plate.
Staining the gel:
1. Place the gel in a small plastic box and cover with the staining solution.
Agitate slowly for 3 h or more on a rotary rocker.
2. Pour off the staining solution and cover the gel with a solution of
destaining solution I. Agitate slowly for 15 min.
3. Pour off the destaining solution I and cover the destaining solution II.
Discard destaining solution and replace with fresh solution. Repeat until
the gel is clear except for the protein bands.