Why do you wish to read A280 for your Cibacron Blue column fractions? You will n

Question

Why do you wish to read A280 for your Cibacron Blue column fractions?

You will need to prepare three buffers to run the CB column. Given the compositions of the buffers and the volumes needed show your calculations needed to prepare them from stock solutions and solid components available.

100 ml of Tris-PMSF Buffer (10 mM Tris-HCl (pH 8.6), 0.5 mM 2-Mercaptoethanol, 1 mM PMSF)

10 ml of NAD Buffer (10 mM Tris-HCl (pH 8.6), 0.5 mM 2-Mercaptoethanol, 1 mM lithium lactate, 1 mM NAD+)

10 ml of NADH buffer (10 mM Tris-HCl (pH 8.6), 0.5 mM 2-Mercaptoethanol, 1 mM NADH)

Stocks:

1.0 M Tris-HCl, pH 8.6
14.3 M 2-mercaptoethanol
100 mM PMSF

solid Lithium lactate (MW = 96.01 g/mol)
solid NAD+ (MW = 663.43 g/mol)
solid NADH (MW = 709.40 g/mol)

Explanation / Answer

Cibacron Blue column is a type of affinity column. As proteins generally has absorbance at 280 nm, thus measuring the absorbance at 280nm will tell whether there is any protein is present or not in the eluent of cibacron clue column.

100 ml of Tris-PMSF Buffer

tris stock= 1.0 M Tris-HCl, pH 8.6

working concenration =10mM)

thus 1M/ 10mM or 1000/10=100 fold dilution is required., thus for 100 ml, 100/100=1 ml is required.

for 100ml we require, 1 ml of Tris-HCl

stock concenraiton of 2-mercaptoethanol=14.3 M

working concentration =0.5 mM

thus 14.3M/ 0.5 mM or 14.3 X 1000/0.5= 28.6 fold diluiton is required.

for 100 ml, 100/ 28.6 or 3.5 ml 2-marceptoehanol

PMSF-stock 100mM

Working concetration= 1mM PMSF

thus 100/1= 100 fold dilution is require.

thus for 100ml 1ml o

f PMSF Is required.

to make 100 ml of Tris-PMSF Buffer, we need

1 ml of Tris-HCl, 3.5 ml 2-marceptoehanol and 1 ml PMSF and 94.5 ml of water.

10 ml of NAD Buffer (10 mM Tris-HCl (pH 8.6), 0.5 mM 2-Mercaptoethanol, 1 mM lithium lactate, 1 mM NAD+)

as 1000ml 1 M lithium lactate =96.01gm

      10ml 1 M lithium acetate contains =96.01X 10/ 1000 =.96 gm

thus 10 ml 1mM lithium acetate contians .96 / 1000=0.00096gm or .96 mg

molecular weight of NAD+ = 663.43 g/mol

1000ml , 1M contains 663.43 g

10ml 1M contains 663.43 X10/1000=6.63gm

10ml, 1mM contains = 66.3 X 1/ 1000=0.0066g or 6.6 mg

thus to make 10 ml of NAD Buffer (10 mM Tris-HCl (pH 8.6), 0.5 mM 2-Mercaptoethanol, 1 mM lithium lactate, 1 mM NAD+) buffer, we require-

1ml of tris-HCl, 3.5 ml of 2--Mercaptoethanl, 0.96 mg of lithium acetate and 6.63 mg NAD+ and 5.5 ml of water.

10 ml of NADH buffer (10 mM Tris-HCl (pH 8.6), 0.5 mM 2-Mercaptoethanol, 1 mM NADH)

solid NADH molecular weight = 709.40 g/mol

thus 1000ml , 1M contains= 709.40gm

10ml, 1M = 709 X 10/ 1000=7.09 gm

10ml , 1mM= 7.09/ 1000=0.007 gm or 7.09 mg

thus 1ml of tris-HCl, 3.5 ml of 2--Mercaptoethanl, 7.09gm NADH and 5.5 ml of water is required.

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