Chromatography

General chromatography: text book experiment 2, beginning on page 25Affinity chromatography: experiment #10, pages 157-162.

from Greek χρῶμα chroma "color" and γράφειν graphein "to write". http://en.wikipedia.org/wiki/Chromatography (12 Jan 2013)

spinach leave pigmentsMobile phase = nail polish remover (acetone, ethyl acetate, butyl acetate),stationary phase = coffee filter paper.

In 9:1 pet ether: acetonecarotene Rf = 0.98xanthophyll Rf = 0.90chlorophyll a Rf = 0.83chlorophyll b Rf = 0.71

http://www.layers-of-learning.com/why-do-leaves-change-color-in-the-fall/

mig

ratio

n

Background! Chromatography is a technique that allows for

the separation of molecules based on their differential migration through a porous medium.

! Molecules partition between the stationary phase and the mobile phase, and this ‘relative mobility’ determines the relative rate of migration.! Rf = (dist. traveled by sample)/(dist. to solv. front)! The closer Rf is to 1, the faster the migration! The closer Rf is to 0, the slower the migration! Rf= 1-α. α is the partition coefficient

• In column chromatography, the stationary phase is a resin.

• The mobile phase is a buffer solution that flows over the resin carrying analyte with it. (More generally, the mobile phase can be mixtures of solvents other than water. Even biomolecules may be separated in water:acetone and water:acetonitrile mixtures.

• The analyte is characterized by the relative extents to which it interacts with the mobile vs. the stationary phases.

[ ] in stationary phase[ ] in stationary and mobile phase

α =

Chromatography video.

http://www.youtube.com/watch?v=9GiLjH9Oym84

elution volume

Abs

orba

nce

many types of chromatography! exploit different interactions.! For purposes of separation, exploit a

feature that distinguishes different compounds present in a mixture, such as size, charge, polarity ...

! For the example of plant pigments the solid phase is cellulose (HCOH)n and the mobile phase is 10% H3COCH3 and 90% light hydrocarbons (pentane-heptane).

5

Rf = 0.98

Rf = 0.90

Rf = 0.83 Rf = 0.71

Chromatography of many kinds

! See tables 2-1, 2-2 in text book! Gel filtration: retardation of large

molecules! Ion-exchange: electrostatic

retention of molecules with a charge opposite to that of the resin.

! Hydrophobic interaction: non-polar surface groups cause molecule to be retained on column (also called reverse phase).

! Affinity: exploits a specific affinity of the molecule for a ligand that can be built into the column or attached to it. (Also requires a release mechanism).

6

Different resins: Size-Exclusion

! Relies on the ability of a given sized molecule to enter the uniformly sized pores of a solid matrix. Molecules that are too big are excluded from the mobile phase within the pores, and exit the matrix faster. Smaller molecules have to go through a larger volume of mobile phase, and so exit later.

! The volume accessible to the molecule dictates its rate of migration.

Ion-Exchange

• Relies on the differential electrostatic affinities of molecules carrying a surface charge for an inert, charged stationary phase.• Cation exchange has negative stationary phase

• Molecules with positive charge will interact with resin.

• Anion exchange has a positive stationary phase• Molecules with negative charge will interact with resin.

• Molecules are eluted by either changing the pH or the ionic strength• pH: the molecule is adsorbed to the column at a

pH that gives it a charge opposite that of the resin, and is eluted by a change in pH that causes it to lose its charge or affinity for the resin.

• Ionic strength: as the concentration of counter-ions in the mobile phase increases, they electrostatically compete with adsorbed molecules bound to the charged resin. Molecules with lower net charge elute with lower salt concentrations.

Ion-Exchange

! Carboxymethyl (CM) cellulose is a cation exchanger

Ion-exchange chromatography

10

!Carboxymethyl (CM) cellulose is a cation exchanger!Diethylaminoethyl (DEAE) cellulose is an anion exchanger

!Elute by increasing ionic strength or by changing pH (chromatofocussing).

http://www.genome.ou.edu/3653/Lecture12-9_18_06

Blue dextran MW = 2,000,000 neutralcytochrome C MW = 12,000 pI = 10.7DNP-glycine MW = 241 pI = 3

resolution on CM sephadex

In ion-exchange mode which should elute first ?In gel filtration mode which should elute first ?

Resolution: separation vs. spreading

! R = 2d /(W1+ W2)

! High-α analytes tend to have more zone spreading (continuously released from the stationary phase into mobile phase passing by).

! A long column is not the solution.! Employ gradient elution instead of

(unchanging) isocratic resolution.11

EXPERIMENT 2 Chromatography 27

Elution volume

d

W1

W2

Con

cent

ratio

n of

com

poun

ds

A B

Figure 2-2 Resolution of two compounds (A andB) emerging from a chromatographycolumn.

areas of high concentration to areas of low con-centration as the mobile phase continuously passesthrough the stationary phase.

Resolution. The goal of any chromatographicstep is to maximize resolution or to minimize zonespreading. Resolution is a function of the positionof the maximum elution peak height and the elu-tion peak width (Fig. 2-2). The greater the resolu-tion between two elution peaks, the greater the de-gree of separation between the two molecules.Mathematically, resolution (R) is two times the dis-tance between two elution peak maxima (d) dividedby the sum of the widths of the two elution peaks(W1 ! W2):

R !

For any given chromatographic step, you mustachieve a compromise between the number of platetransfers and the resolution. In other words, youmust provide enough plate transfers to allow sepa-ration while preventing excess zone spreading.

Isocratic versus Gradient Elution. The principlesof chromatography just described hold true for iso-cratic elution schemes, in which the nature of themobile phase remains constant throughout the de-

2d""(W1 # W2)

velopment of the column. Many chromatographiccolumns, however, are eluted with some sort of mo-bile phase gradient. Here, the nature of the mobilephase changes continuously throughout develop-ment with respect to ionic strength, pH, ligand con-centration, or organic:aqueous solvent ratio. As oneor more of these parameters changes, so too doesthe partition coefficient of one or more moleculesadsorbed on the stationary phase. Although thisgreatly complicates the calculation of $, it cangreatly enhance the ability of a given sized columnto separate a number of different compounds. Bycontinuously changing the partition coefficients ofmolecules passing through the stationary phase, youcan effectively increase the number of plate trans-fers occurring over a given length of the column.A 20-ml column that provides 10 plate transfers uti-lizing an isocratic elution scheme may provide 100to 200 plate transfers when used in conjunction withan elution gradient.

Remember that one problem encountered withincreasing the number of plate transfers is an in-crease in zone spreading, decreasing the resolutionof two elution peaks containing compounds withsimilar partition coefficients. Gradient elutionschemes often work to minimize zone spreading.Recall that molecules with lower $ values show lesszone spreading after a given number of plate trans-fers than molecules with large $ values. If a mole-cule that has a relatively large $ value under load-ing conditions can be eluted with a gradient thatcontinually decreases its $ value as it passes throughthe column, zone spreading can be minimized, andresolution between neighboring elution peaks canbe maintained. In general, mobile phase gradientsare most often used to optimize a specific chro-matographic procedure by maximizing both thenumber of plate transfers and the resolution of theelution peaks.

Types of Chromatography

Gel Filtration or Size Exclusion. Gel filtration isperhaps the easiest type of chromatography to un-derstand conceptually (Fig. 2-3). It relies on theability of a given size molecule to enter the uni-formly sized pores of a solid support or matrix.Imagine that you have a solution containing twomolecules: one of 8000 Da and one of 50,000 Da.If this solution is passed through a porous matrix

Affinity Chromatography

Use a resin that mimics a ligand of the protein.Eg. blue columns in which the resin is decorated with molecules of cibacron blue.

This is mis-recognized as a substrate analog by enzymes that bind NADH and purine nucleotides.Such enzymes bind to the resin more strongly than random proteins do. The latter can be washed away and then the proteins binding the resin can be eluted with authentic substrates, cofactors or analogs (eg. SAM, ATP, GTP, NAD+, NADH, FMN ...

EXPERIMENT 2 Chromatography 37

NH2SO3

!

SO3!

SO3!

N N

ClN

N

N

N

H

H

H

O

O

Figure 2-7 Structure of Cibacron Blue F3GA.

“activate” carbohydrate-based resins for derivatiza-tion with a ligand of interest (Fig. 2-6). Most ofthese chemical reactions take place on the hydroxylgroups of the resin and create intermediates thatare subject to nucleophilic attack by a free aminogroup on the desired ligand.

Dye-Ligand Chromatography. Of all the types ofchromatography presented in this chapter, the prin-ciples governing the technique of dye-ligand chro-matography are perhaps the least well understood.Purely by accident in the 1960s, a blue dye knownas Cibacron Blue was found to have strong affinityfor two enzymes found in yeast: pyruvate kinase and phosphofructokinase. As shown in Fig. 2-7,Cibacron Blue consists of a series of individual andfused aromatic rings containing several conjugateddouble bonds common to molecules that absorblight in the visible range of wavelengths. Ring struc-tures similar to this are present in purine nu-cleotides. This molecule also contains three sul-fonic acid (SO3

!) groups that may be mistaken forphosphate (PO4

!) groups by an enzyme. Based onthese two pieces of evidence, it is currently believedthat Cibacron Blue acts as a substrate analog ofADP-ribose, explaining why the molecule has beenfound to have affinity for many different NAD!-requiring enzymes (dehydrogenases) and kinases(commonly bind purine nucleotides).

Unfortunately, the specificity of this blue dyefor the types of enzymes just described is not asgreat as originally thought. Indeed, Cibacron Bluehas been proven to have great affinity for a num-ber of seemingly unrelated proteins, such as !-interferon and blood serum albumin. The nonspe-cific interactions between the protein and the dyemay be due to cation exchange effects (due to theSO3

" groups) or hydrophobic and/or hydrogen-bonding effects between the proteins and the aro-matic rings on the dye.

A number of methods have proven successful ineluting proteins from Cibacron Blue. Most often,the greatest purification is achieved through somesort of affinity elution scheme. Here, a protein ofinterest bound at its nucleotide binding site to theresin is eluted from it through the addition of itstrue substrate or a high concentration of a substrateanalog to the mobile phase. This free ligand willcompete with the “fixed” groups on the resin forbinding to the protein, lowering its # value. Sub-

strates or ligands commonly used to elute proteinsfrom Cibacron Blue columns include: S-adenosyl-methionine (SAM), ATP, GTP, NAD$, NADH,ADP, AMP, and cAMP. If a suitable ligand cannotbe identified that will cause the protein of interestto elute from the column, elution can be achievedusing the same mobile-phase gradients commonlyused with cation-exchange columns: increasing pHor ionic strength. Two commercially available dye-ligand chromatography adsorbents are shown inTable 2-1.

Immunoaffinity Chromatography. As will be de-scribed in Section IV, an antibody is capable of se-lectively recognizing and binding a single proteinor other antigen in the presence of thousands ofother proteins and other biological molecules. Nat-urally, then, immunoaffinity chromatography offersperhaps the greatest potential in attempting to pu-rify a protein in a single step. A purified antibody(polyclonal or monoclonal) is covalently bound toa stationary-phase resin through which a solutioncontaining a protein of interest is passed. As thecolumn is washed with an appropriate buffer, all ofthe proteins not recognized by the antibody willelute from the column. Ideally, only the protein ofinterest will remain adsorbed to the resin. Unlikethe other types of chromatography discussed thusfar, the challenge in immunoaffinity chromatogra-phy is in trying to determine the conditions of themobile phase that will lower the # value of the pro-tein, causing it to elute from the column.

Antigen–antibody interactions rank as some ofthe strongest found among biological molecules,with dissociation constants (KDs) as high as 10"12 M.

Ni2+-affinity chromatography

! General protocol involves three steps– Bind interesting protein to

chromatography resin.– Wash away all uninteresting proteins.– Elute interesting protein from

chromatography resin (also called matrix).

! We will use a ‘batch’ method rather than a chromatographic application.

! Lower resolution but much faster.! Depends on ability to find conditions

where the target protein binds completely and everything else does not. Modern molecular biology makes this easy ...

13

Ni2+ column IMAC

14

EXPERIMENT 2 Chromatography 39

OCH2

CH2

CH CH

OH

C O!

O

CH2 C O!

O

CH2 C O!

N

Fe3!

Fe3!

Fe3!

Solidsupport

OCH2

CH2

CH CH

OH

C O!

O

CH2 C O!

O

CH2 C O!

N

Ni2!

Ni2!

Ni2!

Solidsupport

6 ! His Protein

Phospho-protein

Figure 2-8 Immobilized metal affinity chromatography resin (nitrilotriacetic acid).

some cases, these types of adsorbents can be de-rivatized with DEAE, polyethyleneimine, acetyl, orC18 groups.

In normal-phase partition chromatography, thestationary phase is polar and the mobile phase usedin development is nonpolar. In reverse-phase par-tition chromatography, the stationary phase is non-polar and the mobile phase used in development ispolar. In thin-layer partition chromatography, thesamples are spotted in a straight line across the bot-tom of the solid support (at the origin). Care mustbe taken to avoid widespread diffusion of these sam-ples (small aliquots of the total applied sample arespotted and allowed to dry). After all the appliedsamples have dried, the plate is placed, origin sidedown, in a chamber containing a small amount ofthe mobile-phase solvent on the bottom. It is es-sential that the origin lies above the level of the mobile-phase solvent in the chamber (see Fig. 6-8)and that the chamber is saturated with the vaporsof the mobile phase before development is begun.

The mobile phases used in the development ofthin-layer chromatography plates are usually com-binations of organic and aqueous solvents. Ethanol,t-amyl alcohol, acetonitrile, and methanol arecommonly used organic solvents. Acetic acid is themost commonly used aqueous solvent. In normal-phase partition chromatography, the polar station-ary phase is developed with relatively nonpolar mo-

bile phase. Compounds will display different ratesof migration through the stationary phase depend-ing on their polarity. More-polar compounds willhave more affinity for the polar stationary phasethan for the nonpolar mobile phase. As a result, themore-polar compounds will travel less distanceacross the plate than the nonpolar compounds,which have a higher affinity for the nonpolar mo-bile phase. The opposite is true for reverse-phasepartition chromatography: the more polar the com-pound, the greater its affinity for the mobile phase,the further that compound will travel across theplate in a given period of time. The behavior of acompound in thin-layer partition chromatographyis usually described in terms of relative mobility(Rf):

Rf "

The larger the value of Rf, the more affinity thecompound has for a particular mobile phase sol-vent.

The major advantages of thin-layer partitionchromatography are cost and versatility. Comparedto column chromatography and HPLC, thin-layerchromatography is very inexpensive to perform. Experimentation with different adsorbents and mobile-phase compositions will allow the separa-

Distance traveled by sample (cm)#####Distance traveled by solvent front (cm)

Figure 2-8

Only proteins that can tightly bind to Ni2+ will be preferentially retained on the column.Such proteins are rare in nature, so a protein engineered to have this capability is generally unique, and the only protein to be retained on such a column.

Qiagen manual “QIAexpressionist.pdf”

15The QIAexpressionist 06/200364

Purif

icatio

n

Native conditions

Celllysis

Denaturing conditions

Ni-NTAresin Bind

Wash

Elute

Pure 6xHis-tagged protein

Tris or phosphatebuffer, pH 8

300 mM NaCl10–20 mM imidazole

Phosphate buffer, pH 88 M urea or 6 M GuHCl(imidazole optional)

30–60 min(Batch or column format)

15–30 min(Batch or column format)

20–50 mM imidazole pH 6.3

pH 5.9 or pH 4.5

100–250 mMimidazole

bla

Figure 20. Purification of 6xHis-tagged proteins using the QIAexpress System.

16

lysa

teflo

w-th

roug

h,el

uate

lysa

teflo

w-th

roug

h,el

uate

llama shark

Single-domain antibodies: promising experimental and therapeutic tools in infection and immunity. Wesolowski et al (2009) Med. Micro. Immun. 198(3).

Encoding affinity for Ni2+

SphI (725)*

BglII (528)*T7 forwardT7

XbaI (462)*NcoI (423)*

HisTEV

NdeI (377)*EcoRI (368)

BamHI (359)*SacI (354)*

AccI (344)XhoI (328)*

T7 reverseT7 term

EcoRV (192)NheI (32)*

AccI (3624)BsmBI (3495)

ApaI (1461)*

BssHII (1661)*EcoRV (1700)

BsmBI (1865)

pBR322 origin

BsaI (4808)*

PstI (4992)*AmpR

AatII (5669)*EcoRI (5740)

pET15TEV_NESG

5741 bp

rbs

Init Sal I XhoI Nco I| Nde I EcoR I BamH I Sac I" HinD III| | TEV Protease | | | | | | | CCATGGGCCATCACCATCACCATCACgaaaacctgtattttcagagcCATATGGCGAATTCTGCGGATCCTGCGAGCTCTGTCGACGCAAAGCTTCTCGAG GGTACCCGGTAGTGGTAGTGGTAGTGcttttggacataaaagtctcgGTATACCGCTTAAGACGCCTAGGACGCTCGAGACAGCTGCGTTTCGAAGAGCTC ______6XHis tag____

M G H H H H H H E N L Y F Q S H M G S A N S A D P A S S V D A K L L

TEV

Normal protein seq.2

Electrophoresis, 12% acrylamide, stained with Coomassie brilliant blue

18

MW

std

ds

Pre

-IPTG

1 hr

. IP

TG

2 hr

. IP

TG

3 hr

. IP

TG

4 hr

. IP

TG

pure

GS

Tfus

ion

www.chembio.uoguelph.ca/educmat/chm357/affinity.pdf

The experiment

1.Use Ni column to purify flavoenzyme.

2.Use SDS-PAGE to visualize the proteins present in different fractions of the column purification.

19

Fractions to save and characterize by SDS PAGE: Cells prior to induction with IPTG*. Crude extract (lysed cells minus debris) Column flow-through Column wash (second one) Eluate

* provided by T.A.

Producing the protein and getting it out of cells

(this will be done for you)! Grow E. coli bearing expression

plasmid to OD600 ≈ 0.5! Add IPTG to initiate expression! Grow 3 hr for protein production! Harvest by centrifugation, discard

supernatant.! Resuspend cell pellet in lysis buffer,

centrifuge down cells again.! Resuspend again in lysis buffer, add

lysozyme (1mg/ml), incubate 30 min. ! Sonicate in bursts to minimize heating.! Centrifuge out debris at 10,000 x g for

20-30 min.

20

The QIAexpressionist 06/2003114

Appe

ndix

Buffers for purification under native conditions (from E. coli and insect cells; protocols 6,9, 11, 12, 13, 14, and 16)

Lysis buffer (1 liter):50 mM NaH2PO4 6.90 g NaH2PO4·H2O (MW 137.99 g/mol)300 mM NaCl 17.54 g NaCl (MW 58.44 g/mol)10 mM imidazole 0.68 g imidazole (MW 68.08 g/mol)Adjust pH to 8.0 using NaOH.

Wash buffer (1 liter):50 mM NaH2PO4 6.90 g NaH2PO4·H2O (MW 137.99 g/mol)300 mM NaCl 17.54 g NaCl (MW 58.44 g/mol)20 mM imidazole 1.36 g imidazole (MW 68.08 g/mol)Adjust pH to 8.0 using NaOH.

Elution buffer (1 liter):50 mM NaH2PO4 6.90 g NaH2PO4·H2O (MW 137.99 g/mol)300 mM NaCl 17.54 g NaCl (MW 58.44 g/mol)250 mM imidazole 17.00 g imidazole (MW 68.08 g/mol)Adjust pH to 8.0 using NaOH.

Buffers for purification from mammalian cells using Ni-NTA Magnetic Agarose Beadsunder native conditions (Protocol 15)PBS 50 mM potassium phosphate, pH 7.2; 150 mM NaClLysis buffer (1 liter):50 mM NaH2PO4 6.90 g NaH2PO4·H2O (MW 137.99 g/mol)300 mM NaCl 17.54 g NaCl (MW 58.44 g/mol)10 mM imidazole 0.68 g imidazole (MW 68.08 g/mol)0.05% Tween 20 5 ml of a 10% Tween 20 stock solutionAdjust pH to 8.0 using NaOH.

prevents non-specific binding

1. Running the Ni column (from the QIAgen manual)

21

Manual purification of 6xHis-tagged proteins (QE06_10_05 Oct-05) page 3 of 7

4. Equilibrate the columns by adding 10 ml Buffer NPI-10 to each column, and apply a vacuum of approximately –10 mbar for 4 min or until buffer has been drawn through.

Avoid allowing the columns to run dry.

5. Transfer the cleared lysates into the equilibrated columns, and apply a vacuum of approximately –10 mbar for 4 min or until buffer has been drawn through.

Avoid allowing the columns to run dry.

6. Perform the first wash step by pipetting 10 ml Buffer NPI-20 into each column, and apply a vacuum of approximately –10 mbar for 4 min or until buffer has been drawn through.

Avoid allowing the columns to run dry.

7. Perform a second wash step by repeating step 6. Very rarely, imidazole concentrations of 20 mM can interfere with binding of 6xHis-tagged

proteins to the resin. If binding is inefficient, reduce the imidazole concentration in the wash buffer (e.g., to 10 mM).

8. Place the 24-well elution vessel inside the QIAvac 6S base. 9. To elute the 6xHis-tagged proteins, pipet 3 ml Buffer NPI-250 into each column, and

apply a vacuum of approximately –10 mbar for approximately 4 min or until buffer has been drawn through.

Avoid allowing the columns to run dry.

Approximately 80% of the bound 6xHis-tagged protein is eluted within the first fraction. If desired, a second elution step can be performed to increase recovery, by repeating step 9. The second eluate can be collected into the same 24-well vessel or into a second 24-well elution vessel.

B: Protein purification under native conditions using gravity flow

Reagents and equipment to be supplied by user

x QIArack (cat. no. 19015)

x Elution vessels (e.g., 4–14 ml polypropylene tubes)

x Buffers NPI-10, NPI-20, and NPI-250

Buffer compositions can be found in the Ni-NTA Superflow BioRobot Handbook, which can be downloaded in convenient PDF format at www.qiagen.com.

1. Position the required number of Ni-NTA Superflow Columns (1.5 ml) on the QIArack. Note: first break the seals at the outlet of the columns before opening the screw cap!

Before use, Ni-NTA Superflow Columns should have been stored in an upright position. Check that the resin is contained in the narrow part of the column body before opening the columns. If the resin is attached to the sides or to the cap of the column, resuspend the resin by inverting the column and allow resin to settle before proceeding with step 2.

2. Remove the storage buffer from above the resin either by using a pipet or by allowing it to drain through by gravity flow.

The columns will not run dry by gravity flow.

3. Equilibrate the columns by pipetting 10 ml Buffer NPI-10 into each column, and allow buffer to drain through completely by gravity flow.

Manual purification of 6xHis-tagged proteins (QE06_10_05 Oct-05) page 4 of 7

4. Transfer the cleared lysates into the equilibrated columns and allow the columns to drain by gravity flow.

5. Perform the first wash step by pipetting 10 ml Buffer NPI-20 into each column. Allow the buffer to drain through completely by gravity flow.

6. Perform a second wash step by repeating step 5. Very rarely, imidazole concentrations of 20 mM can interfere with binding of 6xHis-tagged

proteins to the resin. If binding is inefficient, reduce the imidazole concentration in the wash buffer (e.g., to 10 mM).

7. Place an elution vessel under each column outlet. 8. To elute the 6xHis-tagged proteins, add 3 ml Buffer NPI-250 to each column, allow

buffer to flow through completely, and collect flow-through in the elution vessels. Approximately 80% of the bound 6xHis-tagged protein is eluted within the first fraction. If

desired, a second elution step can be performed to increase recovery, by repeating step 8. The second eluate can be collected into the same or into a second elution vessel.

Protocol 2: Purification of 6xHis-tagged proteins from E. coli under denaturing conditions Under denaturing conditions, the entire lysis and purification procedures should be performed at room temperature (15–25°C).

Reagents and equipment to be supplied by user

x Buffer B-7 M urea

Buffer compositions can be found in the Ni-NTA Superflow BioRobot Handbook, which can be downloaded in convenient PDF format at www.qiagen.com.

Cell lysis and generation of cleared lysates

1. Grow cell cultures, induce protein expression, culture for a previously optimized time period, harvest the cells by centrifugation, and store the pellets at –20°C or at –70°C for at least 1 h.

For details, see the Cultivation of E. coli M15[pREP4] Harboring pQE Expression Constructs protocol in the Ni-NTA Superflow BioRobot Handbook.

2. Place frozen bacterial cells at room temperature and allow to thaw for 15 min. 3. Add 10 ml Buffer B–7 M urea to the thawed cells. In addition, add 3 units Benzonase

for every ml of the original cell culture volume (for example, for a 100 ml cell culture, add 300 units Benzonase).

4. Resuspend the pellet by pipetting up and down. 5. Incubate for 30 min at room temperature. 6. Transfer crude lysate into appropriate tubes and centrifuge for 15 min at 15,000 x g

at room temperature (15–25°C). Insoluble cell components will be pelleted at the bottom of the tube.

7. Collect supernatant containing solubilized 6xHis-tagged proteins and transfer into a fresh tube.

8. Continue with the protein purification by following either procedure A (using the QIAvac 6S) or procedure B (using gravity flow), below.

6xHisTaggedProteins-NiNTAsuperflowColumns.pdfuploaded as:

2. Samples to collect for SDS PAGE

22

Draw arrows into your flow chart showing when these fractions are collected and set aside Cells prior to induction with IPTG*. Crude extract (lysed cells minus debris) Column flow-through Column wash (second one) Eluate

* provided by T.A.

Fractions will be treated with SDS-PAGE sample buffer, denatured by incubation at 90 °C, and run on a polyacrylamide† gel along with molecular weight standards.

† polyacrylamide is a neurotoxin.

SDS PAGE (see experiment 4 in text)

! The text describes the principles and apparatus.

! We will use purchased (precast) gels.! You will simply need to add samples into

the wells, add buffers, hook up to power and supervise.

! Gels will be removed from glass plates and stained with our old friend Coomassie brilliant blue.

! All materials will be made for you. Your execution plan simply needs to provide a sketch of your planned loading arrangement (what samples in which lanes).

! We WILL use the mobilities of molecular weight standards to determine the molecular weight of our test protein.

! (see pages 74-77)23