Size Exclusion Chromatography

II. Procedure Write a Pre-Lab assignment based on the procedure below. This information_ must be in your lab

notebook. Remember to include a title, objective and summarized procedure ,n your own words.

Materials • Bio-Rad glass columns (1.5cm x l0cni)

• Bio-Get• P-100 resin c~1smL)

• lx Phosphate Buffered Saline; PBS (~l00mL)

• Ring stand and clamp ·

• Microcentrifuge tubes; 2ml (~20) , :

• Separation mixture containing ~he fo:llowing compounds (l00µL):

- Blue Oextran (2000 kDa; 3mg/rril; Amax 650nm)

- Myoglobin (16.95 kDa; Smg/ml; Amax 500nm)

- Potassium chromate (194.19 Da; 3mg/ml; Amax440nm)

• BioMate 3S spectrophotometer ·

• Polystyrene cuvettes; 1.5ml (~20)

A. Size-exclusion chromatography

Pour your resin and pack your column:

1. Your resin will be hydrated, de-fined and de-gassed for you. You will begin by setting up and

pouring your column. Make sure the C(?lumn is vertical from side-to-side and front-to-back. The

objective is to produce a uniform _bed o~ the ~ppropriate height with as few additions of slurrv as

possible.

l !

. . erify that the column is

2. Pour some elution buffer (no more than 5ml) Into the col~m; 0

~~ n:t help, discard the buffer

unclogged. If the buffer does not flow out, add some more. If thI5 more buffer through the

and squirt some water into the opposite end of the column. Run some column.

3- Close st0Pcock and add lx PBS buffer until the column is approximately l/3 full.

· fl it is resuspended. Do 4- Resuspend your resin to create a slurry: Gently invert the tube of resm un 1

not shake the tube, as this can damage the beads or introduce air bubbles.

' '

5. Once resuspended, pour the slurry into the ·ccilumn and open the

stopcock at the same time. Fill the column all the way to the top. • I

The flow of buffer through the column will ;,p~ck". the resin. Size­

exclusion columns perform best if packed while the buffer is flowing.

Make sure the stopcock is completely vertical iso the column is open

all the way. Keep adding slurry to the top of the column as it packs.

Do not wait for the column to pack before adding more. ·Rinse the tube

with buffer and add that to the column as well.

6. Once you've added all of your slurry, wait for the resin to pack. Notice

that the packed slurry at the bottom of the column is opaque white,

the resin that is still settling is more translucent, and the buffer

without any resin is clear and colorless (Figure 10), Once your column

is packed, you will only have opaque white resi~ with clear colorless

buffer on top. ·1 !

7. Let most of the buffer drain out into a waste .'beaker. When there is

very little buffer left on top of the resin ("'lmm),.~ close the stopcock.

Equilibrate your column:

Buffer (transparent)

SettllntA!Sin (translucent>

-+-- Settled resin (opaque)

Rgure 10. Settled resin Is opaque. Resin that has not settled yet Is translucent.

8. At this point, you will begin equilibrating your column with two column volumes of fresh PBS • I

buffer. Use a transfer pipette to add buffer to the column. Hold the pipette tip close to the surface

of the resin and very gently add the buffer. Do not disturb the surface of the packed resin. When

you have ~o.Scm of buffer on top of the resin, open the stopcock. Continue adding buffer until it

reaches the top.

***DO NOT LET YOUR COLUMN RUN DRY***

There should ALWAYS be buffer on top of the resin . If the column runs dry, the bed will crack and

the sample will flow through the cracks rather than the beads. You may have to repour the column

and start over. ,, I

9. Continue adding buffer until you have added tv)o column volumes. You do not need to wait for

the buffer level to reach the surface of the res~n \before adding more. I

16

' ,

10. Let the buff . b er dram out until ·t .

uffer so the resin doe , 1 reaches the top of the settled resin . Leave a minimal amount of

sn t run dry• Close the stopcock.

Load your sam I p e and collect fractions:

11. Load lOOµL of th pipette tip t h e_ separation mixture onto the column. Add the sample by gently touching the

edge of th O t _e side of the column just above the resin. Slowly load your sample along the outer

be d ~ resin. If you load your sample unevenly, it will not separate well . Do not disturb the

a s or pierce the resin with the pipette tip.

12. After add' th I

h . mg e sample, open the stopcock. Watch the surface of the resin; when your samp e

as Just entered the resin, close the stopcock.

13· Start collecting fractions now. Position a microcentrifuge tube under your column. Gently add

100µ1 of the elution buffer to the surface of the resin. Be sure to use the same technique you used

to load the sample. Open the stopcock and let it flow out as before. Close the stopcock when the

buffer has just entered the gel. ·

14. Finally, use a transfer pipette to carefully fill the column to the top with elution buffer. D~ not

disturb the resin. Open the stopcock and begin collecting lmL fractions. As you collect fractions,

continue adding buffer to the column to prevent it from going dry.

15. Note that you should begin collecting immediately after loading the sample. How many_ fractions

you should collect is up to your separation; however, you should collect "'2 clear fractions after

the last compound elutes from the column. Keep the fractions in order at all times.

Clean the resin and wash the column:

16. Run several column volumes of buffer through the column to clean the resin. While doing this,

move on to Part B. Keep an eye on yo,ur· column; make sure it doesn't dry out.

17. To empty the column: add some buffer to the column, put a cap on top, make sure the stopcock

is closed, and gently invert. Pour the slurry into the "USED RESIN" beaker so it can be used again

next semester. Do not use anything. otrer than buffer. Add more buffer, resuspend, pour, and

repeat until the column is empty.

18. Rinse the empty column with water. Leave it uncapped to dry.

. Spectrophotometric analysis

You will use a BioMate 3S spectrophotometer to measure the absorbance of each fraction at its ~ax.

1. Organize your fractions into three groups (Blue dextran; blue, myoglobin; red, and potassium

chromate; yellow). Keep your fractions in order. It is often easier to determine which fractions

belong i~ which group if you lay them out next to each other on a white sheet of paper. You may

have a different number of fractions in each group; that is perfectly fine.

2· You will need one cuvette for each fraction, plus one more for the blank. I

17

'th Alconox and water. nless told otherwise. Clean the cuvettes w1 Assume all cuvettes are dirty u b 'th a cotton swa · Rinse thoroughly and dry WI • that appear colorless) r fractions in order (including any 3. Measure the absorbance of all you ·c ATC function. ? according to the steps below. Use the Bas, ( h twill you use for the blank.) a. Set the wavelength to 650nm. Blank the instrument w a b. Measure all fractions in the blue dextran 1group at GSOnm. c. Set the wavelength to 500nm. Blank th~ i_nstrument. d. Measure all fractions in the myoglobin group ~t soonm. e. Set the wavelength to 440nm. Blank th~ instrument. f. Measure all fractions in the potassium chromate group at 44onm.

. ; · h t make sure they are Note: When placing the cuvettes m the spectrop otome er, oriented correctly. Notice that the cuvettes have an inverted triangle alon~ the t~~ edge. Notice also that the cuvette holders inside the instrument have two sides wit holes for the light to pass through. Insert the cuvette such that the triangle on the cuvette is above one of the holes in the holder.

Ill. Data Analysis Before you write your assignment, you must analyze your absorbance vs ml (fraction) data to create a qualitative elution profile of the mixture that you separated by size-exclusion chromatography.

1. Use the absorbance data you recorded in lab~t~ create an elution profile: a. You will need a graphing program (E~cel, Google Sheets, etc) b. Use the Scatter with Smooth Lines and Markers (connect-the-dots) chart type for this plot. c. Present the data as Absorbance (y) vs. ml (x). For this type of qualitative graph, it does not matter that the absorbance values were obtained using more than one wavelength, as this plot is qualitative, not quantitative.

d. The elution peak for each compound should be approximately Gaussian in nature (see Fig. 11).

2. Your elution profile should look similar to Figure 11. The simple title and caption of this figure is just for illustration purposes. Your title must be much more specific and the caption description must be more detailed and thorough.

J

Three Standards u

0.4

G.2

o.o 0 5 10 15

Eluent(ml) 2D

Raure 11. Elution profile of three ~ rds.

IV· Assignment: Figure, Discussion, and Questions

For this assignment, you will create an elution profile and write a Discussion section and References

section. You will also perform a literature search and answer questions regarding different

chromatographic techniques. Below is a guide on how to write these sections. More details on how

to write a lab report are available in your Lab 3 folder on Blackboard. Do not copy and paste the guide

into your document.

1. Figure {20 pts)

Create a figure showing the elution profile of your sample mixture. Be sure to include:

a. proper x- and y-axis labels, including units

b. figure title and caption .

c. Indicate the identity of each peak directly on the graph. You can add text boxes with

information to your chart if using Excel.

d. Include the max absorbance reading and absorbance wavelength for each peak.

2. Discussion (25 pts) : I

Write this section using paragraphs. Pay close attention to the flow of the information presented

in this section. The transitions from point to point should be smooth. Your explanations should

not read like you are answering short questions or bullet points. You will recap your results in this

section. You will also, and most importantly, explain the significance of your results, discuss why

you obtained the results based on previously known information, compare your results to the

literature, explain limitations and importance of study, etc. This section is usually filled with

references.

a. Start the Discussion section with a short introduction stating the purpose of the

experiment and what was done. (No more than 4-5 sentences)

b. Recap your results. Indicate the _ofder in which the mixture components eluted from the

column. Refer to the figure containing your elution profile. Anytime you recap a result in

the discussion, make sure you refer to the figure, table, etc. that you presented in the

Results section. ·

c. Explain the migration rates of e~ch component in the mixture. Explain the order in which

the compounds eluted, taking into. consideration (in no particular order):

• The physical properties of the compound, such as molecular weight

• The nature of the column (what type of chromatography did you use, how is

separation achieved) ' ,

• Exclusion limi~s of the resin. The exclusion limits of a resin indicate the range of

molec~lar we1~hts of the molecules it can separate. This requires that you look up

some mformat1on about the resin you used (Bio-Gel P-100 from Bio-Rad)

• Are these the expected results? If not, explain why.

3. Questions (SOpts total)

Answer the following questions. You do not need to copy and paste th • . .. . e questions m your

assignment. However, number your answfrs. Do not combine answers to different u . .

one paragraph. q est1ons mto

1. Define column volume and void volume What was the column 1 I ? Wh. h · vo ume and void volume of

your co umn 1c compound eluted in the void volume of yo 1 ur co umn? Why? (10 points)

19

. r ted in the table below: 2. Suppose you have a mixture of five proteins ,s

Mol. Weight,

Protein kDa

Answer the following questions (lS points): ; . . 1

f a Bio-Gel p-30 gel-filtration a. Indicate the order in which the prot~ms will e ute rom

column (starting with the one that elutes first). . . ? or wh not? b. Is this the best resin to separate all the components of this ml1lxttuhre. Wmhp~nents yof the

If not, what other Bio-Gel resin can you use to separate a _e co .. . . c. mixture? This requires that you look up information about the size exclusion hmits for

each Bio-Gel resin.

3. You are interested in studying a novel protein (Protein X) discovered in your research lab. You have engineered a version of the protein that contains a 6xHis-tag (hexa histidine). Devise a reliable procedure for its purification from a cell lysate. Answer the following questions. (15

points) a. What is a 6xHis-tag? b. How do you introduce a GxHis-tag to your protein of choice? c. What type of chromatography would you use to purify Protein X? How does it work? d. How you will elute Protein X from the column?

I' i 4. Size-exclusion chromatography is a commt>n technique in biochemical and molecular biology

research labs. Find a research paper that u~es this technique. Describe how it was used in their .study. Use the John Jay Library to conduct your literature search. (10pts)

4. References (lOpts)

You will use references for your literature values and to answer the questions above. When referencing literature for this lab you must:

a. Include at least three references, two of which must be outside references

• Inside references: Any material given to you by the lecture or lab instructor {Biochemistry text book, lab manual, PPT, etc.)

• Outside references: Material not given to you by the lecture or lab instructor (other class textbooks, research papers, reviews, etc.)

b. Use in-text citations (MLA or APA is fine)

5. Observations (-Spts if not included)

You must include a copy for your handwritten b~servations and data collected during lab 1 1 d . , . . nc u e a scan or a picture. Do not re-type your observations.

II. Procedure Write a Pre-Lab assignment based on the procedure below. This information_ must be in your lab

notebook. Remember to include a title, objective and summarized procedure ,n your own words.

Materials • Bio-Rad glass columns (1.5cm x l0cni)

• Bio-Get• P-100 resin c~1smL)

• lx Phosphate Buffered Saline; PBS (~l00mL)

• Ring stand and clamp ·

• Microcentrifuge tubes; 2ml (~20) , :

• Separation mixture containing ~he fo:llowing compounds (l00µL):

- Blue Oextran (2000 kDa; 3mg/rril; Amax 650nm)

- Myoglobin (16.95 kDa; Smg/ml; Amax 500nm)

- Potassium chromate (194.19 Da; 3mg/ml; Amax440nm)

• BioMate 3S spectrophotometer ·

• Polystyrene cuvettes; 1.5ml (~20)

A. Size-exclusion chromatography

Pour your resin and pack your column:

1. Your resin will be hydrated, de-fined and de-gassed for you. You will begin by setting up and

pouring your column. Make sure the C(?lumn is vertical from side-to-side and front-to-back. The

objective is to produce a uniform _bed o~ the ~ppropriate height with as few additions of slurrv as

possible.

l !

. . erify that the column is

2. Pour some elution buffer (no more than 5ml) Into the col~m; 0

~~ n:t help, discard the buffer

unclogged. If the buffer does not flow out, add some more. If thI5 more buffer through the

and squirt some water into the opposite end of the column. Run some column.

3- Close st0Pcock and add lx PBS buffer until the column is approximately l/3 full.

· fl it is resuspended. Do 4- Resuspend your resin to create a slurry: Gently invert the tube of resm un 1

not shake the tube, as this can damage the beads or introduce air bubbles.

' '

5. Once resuspended, pour the slurry into the ·ccilumn and open the

stopcock at the same time. Fill the column all the way to the top. • I

The flow of buffer through the column will ;,p~ck". the resin. Size­

exclusion columns perform best if packed while the buffer is flowing.

Make sure the stopcock is completely vertical iso the column is open

all the way. Keep adding slurry to the top of the column as it packs.

Do not wait for the column to pack before adding more. ·Rinse the tube

with buffer and add that to the column as well.

6. Once you've added all of your slurry, wait for the resin to pack. Notice

that the packed slurry at the bottom of the column is opaque white,

the resin that is still settling is more translucent, and the buffer

without any resin is clear and colorless (Figure 10), Once your column

is packed, you will only have opaque white resi~ with clear colorless

buffer on top. ·1 !

7. Let most of the buffer drain out into a waste .'beaker. When there is

very little buffer left on top of the resin ("'lmm),.~ close the stopcock.

Equilibrate your column:

Buffer (transparent)

SettllntA!Sin (translucent>

-+-- Settled resin (opaque)

Rgure 10. Settled resin Is opaque. Resin that has not settled yet Is translucent.

8. At this point, you will begin equilibrating your column with two column volumes of fresh PBS • I

buffer. Use a transfer pipette to add buffer to the column. Hold the pipette tip close to the surface

of the resin and very gently add the buffer. Do not disturb the surface of the packed resin. When

you have ~o.Scm of buffer on top of the resin, open the stopcock. Continue adding buffer until it

reaches the top.

***DO NOT LET YOUR COLUMN RUN DRY***

There should ALWAYS be buffer on top of the resin . If the column runs dry, the bed will crack and

the sample will flow through the cracks rather than the beads. You may have to repour the column

and start over. ,, I

9. Continue adding buffer until you have added tv)o column volumes. You do not need to wait for

the buffer level to reach the surface of the res~n \before adding more. I

16

' ,

10. Let the buff . b er dram out until ·t .

uffer so the resin doe , 1 reaches the top of the settled resin . Leave a minimal amount of

sn t run dry• Close the stopcock.

Load your sam I p e and collect fractions:

11. Load lOOµL of th pipette tip t h e_ separation mixture onto the column. Add the sample by gently touching the

edge of th O t _e side of the column just above the resin. Slowly load your sample along the outer

be d ~ resin. If you load your sample unevenly, it will not separate well . Do not disturb the

a s or pierce the resin with the pipette tip.

12. After add' th I

h . mg e sample, open the stopcock. Watch the surface of the resin; when your samp e

as Just entered the resin, close the stopcock.

13· Start collecting fractions now. Position a microcentrifuge tube under your column. Gently add

100µ1 of the elution buffer to the surface of the resin. Be sure to use the same technique you used

to load the sample. Open the stopcock and let it flow out as before. Close the stopcock when the

buffer has just entered the gel. ·

14. Finally, use a transfer pipette to carefully fill the column to the top with elution buffer. D~ not

disturb the resin. Open the stopcock and begin collecting lmL fractions. As you collect fractions,

continue adding buffer to the column to prevent it from going dry.

15. Note that you should begin collecting immediately after loading the sample. How many_ fractions

you should collect is up to your separation; however, you should collect "'2 clear fractions after

the last compound elutes from the column. Keep the fractions in order at all times.

Clean the resin and wash the column:

16. Run several column volumes of buffer through the column to clean the resin. While doing this,

move on to Part B. Keep an eye on yo,ur· column; make sure it doesn't dry out.

17. To empty the column: add some buffer to the column, put a cap on top, make sure the stopcock

is closed, and gently invert. Pour the slurry into the "USED RESIN" beaker so it can be used again

next semester. Do not use anything. otrer than buffer. Add more buffer, resuspend, pour, and

repeat until the column is empty.

18. Rinse the empty column with water. Leave it uncapped to dry.

. Spectrophotometric analysis

You will use a BioMate 3S spectrophotometer to measure the absorbance of each fraction at its ~ax.

1. Organize your fractions into three groups (Blue dextran; blue, myoglobin; red, and potassium

chromate; yellow). Keep your fractions in order. It is often easier to determine which fractions

belong i~ which group if you lay them out next to each other on a white sheet of paper. You may

have a different number of fractions in each group; that is perfectly fine.

2· You will need one cuvette for each fraction, plus one more for the blank. I

17

'th Alconox and water. nless told otherwise. Clean the cuvettes w1 Assume all cuvettes are dirty u b 'th a cotton swa · Rinse thoroughly and dry WI • that appear colorless) r fractions in order (including any 3. Measure the absorbance of all you ·c ATC function. ? according to the steps below. Use the Bas, ( h twill you use for the blank.) a. Set the wavelength to 650nm. Blank the instrument w a b. Measure all fractions in the blue dextran 1group at GSOnm. c. Set the wavelength to 500nm. Blank th~ i_nstrument. d. Measure all fractions in the myoglobin group ~t soonm. e. Set the wavelength to 440nm. Blank th~ instrument. f. Measure all fractions in the potassium chromate group at 44onm.

. ; · h t make sure they are Note: When placing the cuvettes m the spectrop otome er, oriented correctly. Notice that the cuvettes have an inverted triangle alon~ the t~~ edge. Notice also that the cuvette holders inside the instrument have two sides wit holes for the light to pass through. Insert the cuvette such that the triangle on the cuvette is above one of the holes in the holder.

Ill. Data Analysis Before you write your assignment, you must analyze your absorbance vs ml (fraction) data to create a qualitative elution profile of the mixture that you separated by size-exclusion chromatography.

1. Use the absorbance data you recorded in lab~t~ create an elution profile: a. You will need a graphing program (E~cel, Google Sheets, etc) b. Use the Scatter with Smooth Lines and Markers (connect-the-dots) chart type for this plot. c. Present the data as Absorbance (y) vs. ml (x). For this type of qualitative graph, it does not matter that the absorbance values were obtained using more than one wavelength, as this plot is qualitative, not quantitative.

d. The elution peak for each compound should be approximately Gaussian in nature (see Fig. 11).

2. Your elution profile should look similar to Figure 11. The simple title and caption of this figure is just for illustration purposes. Your title must be much more specific and the caption description must be more detailed and thorough.

J

Three Standards u

0.4

G.2

o.o 0 5 10 15

Eluent(ml) 2D

Raure 11. Elution profile of three ~ rds.

IV· Assignment: Figure, Discussion, and Questions

For this assignment, you will create an elution profile and write a Discussion section and References

section. You will also perform a literature search and answer questions regarding different

chromatographic techniques. Below is a guide on how to write these sections. More details on how

to write a lab report are available in your Lab 3 folder on Blackboard. Do not copy and paste the guide

into your document.

1. Figure {20 pts)

Create a figure showing the elution profile of your sample mixture. Be sure to include:

a. proper x- and y-axis labels, including units

b. figure title and caption .

c. Indicate the identity of each peak directly on the graph. You can add text boxes with

information to your chart if using Excel.

d. Include the max absorbance reading and absorbance wavelength for each peak.

2. Discussion (25 pts) : I

Write this section using paragraphs. Pay close attention to the flow of the information presented

in this section. The transitions from point to point should be smooth. Your explanations should

not read like you are answering short questions or bullet points. You will recap your results in this

section. You will also, and most importantly, explain the significance of your results, discuss why

you obtained the results based on previously known information, compare your results to the

literature, explain limitations and importance of study, etc. This section is usually filled with

references.

a. Start the Discussion section with a short introduction stating the purpose of the

experiment and what was done. (No more than 4-5 sentences)

b. Recap your results. Indicate the _ofder in which the mixture components eluted from the

column. Refer to the figure containing your elution profile. Anytime you recap a result in

the discussion, make sure you refer to the figure, table, etc. that you presented in the

Results section. ·

c. Explain the migration rates of e~ch component in the mixture. Explain the order in which

the compounds eluted, taking into. consideration (in no particular order):

• The physical properties of the compound, such as molecular weight

• The nature of the column (what type of chromatography did you use, how is

separation achieved) ' ,

• Exclusion limi~s of the resin. The exclusion limits of a resin indicate the range of

molec~lar we1~hts of the molecules it can separate. This requires that you look up

some mformat1on about the resin you used (Bio-Gel P-100 from Bio-Rad)

• Are these the expected results? If not, explain why.

3. Questions (SOpts total)

Answer the following questions. You do not need to copy and paste th • . .. . e questions m your

assignment. However, number your answfrs. Do not combine answers to different u . .

one paragraph. q est1ons mto

1. Define column volume and void volume What was the column 1 I ? Wh. h · vo ume and void volume of

your co umn 1c compound eluted in the void volume of yo 1 ur co umn? Why? (10 points)

19

. r ted in the table below: 2. Suppose you have a mixture of five proteins ,s

Mol. Weight,

Protein kDa

Answer the following questions (lS points): ; . . 1

f a Bio-Gel p-30 gel-filtration a. Indicate the order in which the prot~ms will e ute rom

column (starting with the one that elutes first). . . ? or wh not? b. Is this the best resin to separate all the components of this ml1lxttuhre. Wmhp~nents yof the

If not, what other Bio-Gel resin can you use to separate a _e co .. . . c. mixture? This requires that you look up information about the size exclusion hmits for

each Bio-Gel resin.

3. You are interested in studying a novel protein (Protein X) discovered in your research lab. You have engineered a version of the protein that contains a 6xHis-tag (hexa histidine). Devise a reliable procedure for its purification from a cell lysate. Answer the following questions. (15

points) a. What is a 6xHis-tag? b. How do you introduce a GxHis-tag to your protein of choice? c. What type of chromatography would you use to purify Protein X? How does it work? d. How you will elute Protein X from the column?

I' i 4. Size-exclusion chromatography is a commt>n technique in biochemical and molecular biology

research labs. Find a research paper that u~es this technique. Describe how it was used in their .study. Use the John Jay Library to conduct your literature search. (10pts)

4. References (lOpts)

You will use references for your literature values and to answer the questions above. When referencing literature for this lab you must:

a. Include at least three references, two of which must be outside references

• Inside references: Any material given to you by the lecture or lab instructor {Biochemistry text book, lab manual, PPT, etc.)

• Outside references: Material not given to you by the lecture or lab instructor (other class textbooks, research papers, reviews, etc.)

b. Use in-text citations (MLA or APA is fine)

5. Observations (-Spts if not included)

You must include a copy for your handwritten b~servations and data collected during lab 1 1 d . , . . nc u e a scan or a picture. Do not re-type your observations.