Dear Megan and Lan,

As you are aware, we will be doing some protein purifications and also protein expression in E. coli
cells.

Due to time constraints (around your exams and my own constraints), we will have to purify the
protein starting on Monday 6th. If time allows, we can also do the step prior to this where we grow
the E. coli and express the proteins. I will already have the necessary E. coli cells ready for you to
purify from Monday onwards.

The protein is a 6xHis-tag protein, which will bind to Ni-NTA resin. We then wash off other proteins
that do not bind to the resin, and subsequently elute off the protein using high concentrations of
imidazole (see if you can find out why high imidazole will elute the protein). Once eluted, we need
to desalt the protein into a buffer of our choice (without imidazole as it inhibits a protein called
thrombin which we need to use to remove the 6xHis-tag). Once desalted, we will add the thrombin
to cleave the 6xHis-tag overnight. Then we will run the protein over a size exclusion column (see if
you can find out the principle of how these work).

To do the purifications we need a certain set of buffers that get used at different stages of the
purification. Although I can do all of these calculations to make the buffers, it is good practice for
you to do it as well, as these are necessary skills to work efficiently in a laboratory.

The following exercise will require you to do a bit of internet searching to find out information online
that is required for you to do the calculations.

a) For each buffer I would like you to work how much of each stock solution is required to
produce the buffer at the final required concentrations.
b) Work out the mass of each component that would have to be dissolved to make the
required volume of buffer stated.

Here is an example.

Buffer A – 1L required
30 mM KOAc
100 mM RbCl
10 mM CaCl2
50 mM MnCl2
3 mM CoCl2.6H2O
15% (v/v) Glycerol

Method 1: From stock solutions.

Stock solution – volume required

1M KOAc – 30 mL
2M RbCl – 50 mL
1M CaCl2 – 10 mL
1M MnCl2 – 50 mL
0.5M CoCl2.6H2O – 6 mL
100% (v/v) Glycerol – 150 mL
Add Acetic acid or KOH to pH to 6.5
Add deionised water to 1L
Equations used: 𝐶𝑠 × 𝑉𝑟 = 𝐶𝑟 × 𝑉𝑡

Cs = concentration of stock solution

Vr = Volume of stock solution required
Cr = Concentration required
Vt = Total volume

Method 2: Dissolve solid components.

Buffer component Molecular weight (Da) Mass required (g)

KOAc 98.14 2.9442
RbCl 120.92 12.092
CaCl2 110.98 1.1098
MnCl2 6.292
CoCl2.6H2O 237.93 0.71379
Glycerol N/A - this is a liquid 150 mL, NOT 150 g

Add all solids to a beaker. Add minimal amount of deionised water and the required glycerol to
dissolve the solids. Add Acetic acid or KOH to pH to 6.5. Add deionised water to 1L.

Equations used:
𝑀𝑎𝑠𝑠 (𝑔)
𝑚𝑜𝑙𝑒𝑠 (𝑚𝑜𝑙) =
𝑀𝑜𝑙𝑒𝑐𝑢𝑙𝑎𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 (𝐷𝑎)
𝑚𝑜𝑙𝑒𝑠 (𝑚𝑜𝑙)
𝑀𝑜𝑙𝑎𝑟 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑀) =
𝑣𝑜𝑙𝑢𝑚𝑒 (𝐿)

That’s the example. These are the buffers that we are very likely to use and I would like you to
calculate using both methods shown above. You will realise the advantage of using method 1 as it is
far more practical in day-to-day use.

Lysis buffer + protease inhibitor– 50 mL required

50 mM Tris-HCl pH 8.0
500 mM NaCl
10 mM Imidazole pH 8.0
2 mM DTT (dithiothreitol)
0.1 % (v/v) Tween 20
1 tablet of EDTA-free protease inhibitor (bonus marks if you can tell me why EDTA-free is required).

Lysis buffer (no protease inhibitor) – 50 mL required

50 mM Tris-HCl pH 8.0
500 mM NaCl
10 mM Imidazole pH 8.0
2 mM DTT
0.1 % (v/v) Tween 20
Wash buffer – 500 mL required
50 mM Tris-HCl pH 8.0
500 mM NaCl
10 mM Imidazole pH 8.0
2 mM DTT

Elution buffer I – 30 mL required

50 mM Tris-HCl pH 8.0
500 mM NaCl
250 mM Imidazole pH 8.0
2 mM DTT

Elution buffer II – 30 mL required

50 mM Tris-HCl pH 8.0
500 mM NaCl
500 mM Imidazole pH 8.0
2 mM DTT

Elution buffer III – 30 mL required

50 mM Tris-HCl pH 8.0
500 mM NaCl
1 M Imidazole pH 8.0
2 mM DTT

Desalting buffer – 250 mL required

50 mM Tris-HCl pH 8.0
300 mM NaCl
2 mM DTT

Size exclusion chromatography buffer (2L required)

50 mM HEPES (free acid) pH 7.5
300 mM NaCl
2 mM DTT

Good luck and if you have any questions or you get stuck/ don’t understand something just ask!

My email address is rse27@cam.ac.uk

Best,
Rohan