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Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. 1.Ni-column electrophoresis (already discussed in lecture) 2.Sodium dodecyl sulfate - polyacrylamide gel electrophoresis = SDS PAGE (today’s lecture). 1

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Page 1: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

Affinity Chromatography and SDS-PAGE

SDS PAGE: Experiment #4 of text.

1.Ni-column electrophoresis (already discussed in lecture)

2.Sodium dodecyl sulfate - polyacrylamide gel electrophoresis = SDS PAGE (today’s lecture).

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Page 2: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

Electrophoresisl Electrophoresis is the process whereby

charged molecules migrate through a solution under the influence of an applied electric field.

l In gel electrophoresis, a gel matrix of an aqueous solution is subjected to an applied electric field. Samples are loaded into one end of the gel, and ‘pulled through the matrix by the attraction between their charge and the oppositely-charged electrode.

l The sample molecules must have charge. DNA and RNA have negative charge and the charge to mass ratio is constant: one negative per based. Nucleic acids therefore migrate towards positive charge.

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Page 3: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

l There are two common types of gel electrophoresis: those based on an agarose gel matrix, and those based on a polyacrylamide gel matrix.

l Polyacrylamide gels can give much greater resolution than agarose gels, but are much more cumbersome to make.

l Protein separations typically use polyacrylamide gels. Nucleic acid separations typically use agarose for low resolution and polyacrylamide for high resolution separations.

l Effective pore size of the matrix can be manipulated by altering the degree of crosslinking within these gels. This is done to achieve the desired separation range.

Background

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Page 4: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

Separation Ranges

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Page 5: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

Electrophoresisl Under native conditions, a protein can have

a positive or a negative charge, which can be large or small. To eliminate this source of variability, the anionic detergent SDS (sodium dodecyl sulfate) is used to fully coat the proteins in a sample. Now every protein has a relatively uniform charge-to-mass ratio.

l Proteins also have differing shapes and rigidities, they can also be constrained by disulfide bonds or complexed together in large oligomers. To eliminate these differences between proteins that mask their true molecular weights we denature them using heat, the SDS and β-mercaptoethanol. Now they will experience resistance from the polyacrylamide gel simply in proportion to their size, and can be separated (resolved) on that basis.

l SDS-PAGE is described on pages 65-71.

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Page 6: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

The effect of SDS and β-mercaptoethanol on

proteins

l ≈ 1 dodecyl sulfate / 2 amino acids

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Page 7: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

Relative mobility in PAGE• The mobility of a molecule increases with increasing

applied voltage, increasing net charge on the molecule, and decreasing friction of the molecule.

• mobility = (voltage x charge)/(friction)

• Hence, information on SIZE.• A MW ‘standard’ curve is created by simultaneously

running proteins of known MW. Plot log(MW) vs. Rf.

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Page 8: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

How stacking gels improve resolution.

l At pH 8.3 glycine has a charge of -1, but at pH 6.8 it is neutral (slow).

l Dodecyl sulfate has a charge of -1 at both pHs, so does Cl-.

l Glycine is very small (fast when charged), but the proteins are big (slow).

l The resolving gel has a smaller pore size and more greatly restrains the proteins.

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Glycine

Cl-

Cl-Glycine

Glycine

Cl-

GlycineCl-

Page 9: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

SDS-PAGESetting up the electric field.

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Page 10: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

SDS-PAGE, visualizationl Dyes are incorporated with samples to

analyze gel migration properties.– Is the gel working?– How far have the sample run?

l After the gel is run, two methods are used to visualize the individual protein banding patterns.

l Acetic acid and methanol precipitate proteins and ‘fix’ them in place.

– Coomassie Brilliant Blue R250 binds non-specifically to all proteins, turning protein bands blue. Binds to Arg (and Trp, Tyr, Phe, His & Lys). 0.1 - 0.5 µg protein can be detected.

– Alternately you can stain with substance that specifically binds a particular protein, and on binding somehow generates a visual signal. l A western blot is when antibodies are used

to specifically bind a protein, which then is used to generate a visual signal

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Page 11: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

E. coli total cell protein

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Figure: SDS-PAGE gel (stained with Coomassie blue) showing

overexpression of SOD2-pEL002 and SOD-pET23d. Lane 1, prestained SDS-

PAGE standards, low range; lane 2, SOD2-pEL002 cells without addition of

IPTG; lane 3, SOD2-pEL002 cells with addition of IPTG; lane 4, SOD-pET23d

cells without addition of IPTG; lane 5, SOD-pET23d cells with addition of

IPTG; lane 6, kaleidoscope polypeptide standards.

Protein over-expression upon

induction with IPTG

More modest overexpression from a different construct.

Elec

trop

hore

sis

small proteins

big proteins

Page 12: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

The Experiment

l We will isolate His-tagged nitroreductase, using immobilized metal ion affinity chromatography (Ni column).

l Day 1: Run Ni column.

l Day 2: Analyze fractions saved from IMAC by SDS PAGE.

Page 13: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

Day 1l Obtain cell extract from T.A.l Isolate His-tagged nitroreductase from the

lisate, using IMAC over a Ni column (protocol follows).

l Save fractions: Crude extract, Column flow-through, Column wash (second one), Eluate.

l Questions for your post-lab write-up:l Could there be non-specific binding to the Ni

column (proteins other than the His-tagged nitroreductase) ?

l If so, what properties would proteins have to have in order to bind the column.

l How can we separate non-specifically bound proteins from His-tagged nitroreductase ?

l What could cause an IMAC column to not function ?

l Name two ways in which you could dislodge His-tagged nitroreductase from the column.

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Page 14: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

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

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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 to web site as:

Page 15: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

Day 2: SDS PAGE (see experiment 4 in text)

l The text describes the principles and apparatus.

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

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

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

l 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).

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

l Also DO exercises 1 and 2 (pages 74-77)

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Page 16: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

Changes From the Bookl In your prelab, sketch what you expect

your gel to look like, assuming staining with Coomassie brilliant blue, which detects all proteins. Don’t forget that you will have the samples you made on Tuesday PLUS a sample of cells harvested before induction with IPTG, and molecular weight standards.

l The basic procedure of running SDS gels is outlined in experiment #4. Running at 200 V increases the migration speeds.

l Skip steps 1-7. Assemble the running assembly as instructed by T.A. (approximately as in Figure 4-10).

l In step 9, the concentrated protein sample buffer is 2x, not 4 x. Therefore, use 10 µl of a protein sample (from Tuesday) + 10 µl of 2x protein sample buffer.

l We will load our gels using pipetmen not syringes.

l Our Molecular Weight standards are from Biorad, use 10 µl MW standards without dilution (Biorad part #161-0324).

Page 17: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

Pictures

Page 18: Affinity Chromatography and SDS-PAGE - University of · PDF file · 2013-02-25Affinity Chromatography and SDS-PAGE SDS PAGE: Experiment #4 of text. ... using immobilized metal ion

Safety Considerationsl Observe all normal laboratory safety

practices.

l Dispose of all waste into the proper waste containers.

l Be cautious around electricity.

l Acrylamide is a neurotoxin !!

l Are ALL the compounds we will use familiar to you with respect to their safety implications ?

IPTG

Acrylamide ?