Precipitation

Ammonium sulphate salt is used as precipitate in the first precipitation step followed
by Magnesium chloride in 2nd precipitation.

Purpose and expected output

Used as an initial capturing step . Proteins bind to each other due to salting out.
Removes soluble protein and other proteins and hopefully DNA.

Equipments
Small scale- falcon tubes
Large scale- tanks up to 100 to 300 liters

Materials
Ammonium sulphate - (NH₄)₂SO₄
Magnesium chloride - MgCl₂
base- Ammonium hydroxide
acid- sulphuric acid

Protocol for Precipitation with pure protein sample

● Measure the pure protein concentration at 280 nm and enzyme activity (using ONPG
assay) of the given pure sample
● Perform dilution if the enzyme is highly concentrated - to measure in the linear range
of the assay

1. Testing Ammonium sulphate precipitation for pure sample

1. Take 10 ml of pure sample with constant beta-gal concentration in a 15 ml
falcon tube
2. Add required amount of Ammonium sulfate (table 1 ) for the different desired
saturation (20% to 90%) to the sample by weighing (w/v %)
3. Check the pH in between - maintain at pH 7
4. Gentle stirring at 4oC room in an inverting rotator for 1 hour0
5. Then centrifuge the sample at 13000 rpm for 15 mins at 4oC and collect the
pellet
6. Resuspend the pellet in 10 ml of buffer (0.1 M MOPS)
After precipitation:
● Measure the protein concentration at A280 nm and enzyme activity (using ONPG
assay) of the given sample
● Measure the DNA concentration at A260 nm

Table 1 Different saturation % and corresponding ammonium sulfate required

Saturation % Volume of pure sample Ammonium sulphate (g)
(ml)

20 10 1.1

30 10 1.7

40 10 2.33

50 10 3.01

60 10 3.73

70 10 4.5

80 10 5.33

90 10 6.21

2. Testing Magnesium chloride precipitation for pure sample

1. Add 1 ml of 1M MgCl2 solution to 9 ml of sample (for 0.1M mgcl2 concentration)

in a tube and add 2 ml 1M MgCl2 solution to 8 ml of sample (for 0.2M mgcl2
concentration)
2. Check the pH and maintain at pH 7
3. Gentle stirring at 4oC room in an inverting rotator for 1 hour
4. Then centrifuge the sample at 13000 rpm for 15 mins 4oC and collect the pellet
5. Resuspend the pellet in 10 ml of buffer (0.1 M-MOPS)

After precipitation:
● Measure the protein concentration at A280 nm and enzyme activity (using ONPG
assay) of the given sample
● Measure the DNA concentration at A260 nm
For Batch process sample - week 3 on thursday

Summary of two-step precipitation

Exp precipitation by Analysis

1. Ammonium sulphate and then ONPG , A260 (DNA), A280

magnesium chloride (Protein) before, in between and
after precipitation

2 Magnesium chloride ONPG , A260 (DNA), A280

(Protein) before and after

1.Testing Ammonium sulphate precipitation for lysate sample

Before precipitation:
● Measure the protein concentration at A280 nm and enzyme activity (using ONPG
assay) of the given sample
● Measure the DNA concentration at A260 nm

Precipitation step with Ammonium sulphate

1.Take 10 ml of cell lysate in a falcon tube
2. Add the amount of ammonium sulphate ( 30% to 70%) to the sample (ref table 2)
(w/v %)
3. Check the pH in between - maintain at pH 7
4. Gentle stirring at 4oC room in an inverting rotator for 1 hour
5. Then centrifuge the sample at 13000 rpm for 15 mins at 4oC and collect the pellet
6. Resuspend the pellet in 10 ml of buffer (0.1 M MOPS)

After precipitation:
● Measure the protein concentration at A280 nm and enzyme activity (using ONPG
assay) of the given sample
● Measure the DNA concentration at A260 nm
Table 2 Different saturation % and corresponding ammonium sulfate required
Saturation % Volume of pure sample Ammonium sulphate (g)
(ml)

30 10 1.7

40 10 2.33

50 10 3.01

60 10 3.73

70 10 4.5

2.Testing Magnesium chloride precipitation for lysate sample

Before precipitation:
Measure the protein concentration at 280 nm and enzyme activity (using ONPG assay) of the
given sample
● Perform dilution if the enzyme is highly concentrated
● Measure the A260/A280 to determine amount of DNA and RNA in sample

1. Add 1 ml of 1M MgCl2 solution to 9 ml of lysate sample (for 0.1M mgcl2 concentration)

in a tube and add 2 ml 1M) to 10 ml of pu sample and add 2 ml 1M MgCl2 solution to 8 ml
of sample (for 0.2M mgcl2 concentration)
2. Check the pH in between - maintain at 7
3. Gentle stirring at 4oC room in an inverting rotator for 1 hour
4. Then centrifuge the sample at 13000 rpm for 15 mins at 4oC and collect the pellet
5. Resuspend the pellet in 10 ml of buffer (0.1 M MOPS)

After precipitation:
● Measure the protein concentration at A280 nm and enzyme activity (using ONPG
assay) of the given sample
● Measure the DNA concentration at A260 nm
3. Testing Ammonium sulphate + Magnesium chloride precipitation
for lysate sample

a) First Precipitation step with Ammonium sulphate

1.Take 10 ml of cell lysate in a falcon tube
2. Add the optimized amount of ammonium sulphate to the sample (w/v %)
3. Check the pH in between - maintain at pH 7
4. Gentle stirring at 4oC room in an inverting rotator for 1 hour
5. Then centrifuge the sample at 13000 rpm for 15 mins at 4oC and collect the pellet
6. Resuspend the pellet in 10 ml of buffer (0.1 M MOPS)

b) Second precipitation step with magnesium chloride

8. Take the remaining resuspended pellet sample from step a) 6 and add optimized
concentration of magnesium chloride (v/v %)
9. Check the pH in between - maintain at pH 7
10. Gentle stirring at 4oC room in an inverting rotator for 1 hour
11. Then centrifuge the sample at 13000 rpm for 15 mins at 4oC and collect the pellet
12. Resuspend the pellet in 10 ml of buffer (0.1 M MOPS)

After precipitation:
● Measure the protein concentration at A280 nm and enzyme activity (using ONPG
assay) of the given sample
● Measure the DNA concentration at A260 nm