07NOV17 IRM 12 protein purification

- I have made 0.5 lt of 1M of MES buffer adjusted to pH 6.5 for IRM12 and 0.5lt of 1M Tris adjusted to pH 9.0 for IRM13.

The pI of the proteins is IRM12: 5.64 and IMR13: 8.52 but especially for the IRM12 we used the pH 6.5 because acidic conditions might cause proteins to precipitated and make assemblies (for sonication or for non clear extracts). Its not a problem to use 1-1.5 pH more or less than the pI of the protein

-I also made the A buffer and the B buffer containing either MES or Tris (depending on the protein). Everything that has Imidazole is stored in the fridge.

Today I am going to purify starting with the IRM12 - EVERYTHING ON THE ICE

SONICATION with MES

- I thaw 2.14gr of biomass which means I need 2.14 x 5 = 10.7 ml of sonication buffer. The sonication buffer will contain CHAPS, NaCl, PMSF, benzonase and Glycerol made up to the required volume with MES 50mM (see page 116 in DR 0817 - 073)

- Sonication using the large probe in 28% amplitude for 7.5 minutes (these factors are adjusted depending on the sound of the sonicator and signs of sonicating).

- In the meantime, I wash and equilibrate the resin. I need to get rid off the ethanol and also to equilibrate it to the pH I going to use. I am going to use 0.3 x CFE which means about 0.3 x 11ml = 3.3ml resin and spin the falcon down for 5min at 3900rpm. Wash the resin with 3 x resin volume = 9.9ml H2O first for 2 times. Then I wash the resin with 2 x resin volume  = 6.6ml of buffer A.

- I spin down the lysate at 4oC at 10,000rpm for 30 minutes

- Collect the supernatant and resuspend the pellet with the same volume of sonication buffer I used but H2O ie. 10.7ml (this will be our soluble and insoluble fraction). I take a sample of the soluble and insoluble fraction for running on the gel. Incubate the soluble fraction with the resin on the rocker in the cold room for 2 hours.

- Then I set the column. I need a stand with a clamp. I clamp the tip and adjust the fennel. I spill the CFE (soluble fraction) in the fennel and collect the flow through (Non Binding, NB) in a falcon  in a bucket of ice. I can help the collection by pushing with my hand the top of the fennel. I collect the non binding phase first.

I made 50mM, 150mM, 250mM and 350mM Imidazole concentration by diluting buffer B with buffer A. First I calculate how much of the buffer A I need to add to the buffer B to make 10ml of buffers with the required concentration (C1V1 = C2V2).

For making:

10 ml of 50mM Imidazole buffer I need 9ml buffer A and 1ml of buffer B
150mM Imidazole buffer I need 3ml buffer A and 7ml of buffer B

250mM Imidazole buffer I need 5ml buffer A and 5ml of buffer B

350mM Imidazole buffer I need 7ml buffer A and 3ml of buffer B

Consequently the NaCl concentation is changing as follows:

50mM Imidazole buffer has 465mM NaCl
150mM Imidazole buffer has 395mM NaCl

250mM Imidazole buffer has 325mM NaCl

350mM Imidazole buffer has 255mM NaCl

I calculate this by adding the concentration of NaCl in buffer A and in buffer B. For example if I need 1ml of buffer B to be diluted with 9ml of buffer A I have:

(150mM x 1ml)/10ml = 15 mM for buffer B
(500mM x 9ml)/10ml =  450 mM for buffer A

In the final buffer with 50mM imidazole concentration we will have 450 + 15 = 465mM NaCl

 I follow the instructions in page see page 116 in DR 0817 - 073

DISCREPANCY FROM THE PROTOCOL
I used

3 x resin volume = 11ml of 50mM MES, NaCl and 50mM Imidazole
3 x resin volume = 11ml of 50mM MES, NaCl and 150mM Imidazole
2 x resin volume = 6.6ml of 50mM MES, NaCl and 250mM Imidazole
2 x resin volume = 6.6ml of 50mM MES, NaCl and 350mM Imidazole
2 x resin volume = 6.6ml of 50mM MES, NaCl and 500mM Imidazole x 2 times

I make the washes and the elutions passing the solutions each time once but for the elution phase with the 500mM Imidazole solution I made two washes.

I kelp everything and took 10μl of every sample to prepare it with SDS sample buffer, DTT 9% and H2O for running on an SDS page. I run them like this:

INSOL SOL NB 50mM 150mM 250mM 350mM 500mM 500mM

For the INSOL, SOL and NB I loaded 10μl on the gel while for all the others I loaded 20μl on the gel.

I left the gel to stain overnight while I kept the fractions in the freezer