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Preparation Of SDS: Preparation Of GES

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SDS – PAGE (10%)
PREPARATION OF GELS:
RESOLVING GEL PREPARATION:
1. Pipette out 2.66 ml of 30% Acrylamide and pour in beaker.
2. Add 2 ml of Lower Tris (L-Tris, 1.5 M, 8.8 pH) to this.
3. Take 1.96 ml distilled H2O and pour it into this beaker.
4. Now take 1.2 ml Glycerol and add to this mix.
5. Pipette out 80 microliter of 10% SDS (Sodium Dodecyl Sulfate) and pour into the beaker.
6. Add 80 microliter APS (Ammonium per sulfate) as well as 8 microliter TEMED to this as well.
7. Pipette out 4 microliter of TEMED and add it to the beaker as well.
8. Mix these contents together properly and resolving gel (lower gel) is prepared.
CASTING GEL PREPARATION: (3 ml)
1. Pipette out 0.3 ml of 30% Acrylamide and pour in beaker.
2. Add 0.75 ml Upper Tris
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 After polymerization of lower gel, now add the Casting Gel and keep it undisturbed for around an hour, so that it can solidify.
 After this, sample preparation is done. Add sample buffer to samples in 3:1 ratio. The Sample Buffer is made of SDS, BPB and Beta Mercaptoethanol.
(SDS denatures the proteins, BPB is used as a tracking dye while Beta Mercaptoethanol breaks sulfide bonds.)
 Heat the samples at 80 degrees Celsius for 5 minutes in a water bath. This helps in protein denaturation.
 Put the cassette into the buffer dam and fill it with the running buffer. For SDS-PAGE, running buffer is prepared by taking 40 ml Tris-Glycine and adding 4 ml of 10% SDS to it. Its volume is raised up to 400 ml by adding distilled water.
 Load these samples into wells created in the gel.
 Run the electrophoresis unit at 5 mA and 20 V for entry of sample into lower gel.
 Once the samples enter the lower gel, alter the current to 10 mA and set the voltage at 90-100 V.
 Leave this setup undisturbed for around 3 hours.
 Once the gel has run and the samples have reached the bottom end of the cassette (a faint blue line is visible), the gel can be taken
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Only a limited sample analysis can be done using electrophoresis as this technique is quite specific in nature. Therefore, at a time, electrophoresis can only be used for analyzing samples obtained from some particular or specific tissues, and from nowhere else. This specificity results in accurate results, but can be detrimental in cases where researchers are interested in more widespread studies or results.
3. Measurements obtained through electrophoresis are not always precise. Though gel electrophoresis is very effective in separating similar proteins with different molecular weights, and nowadays it uses 2 dimensional techniques to obtain further precision, most measurements made by this technique can be considered semi-quantitative at best. Even today, if obtainment of precise mass (weight) of proteins is desired, mass spectroscopy must be carried out after the protein has been purified by electrophoresis.
Furthermore, random errors are quite rampant and samples are usually run multiple times to get clean and correct
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