Multi-column purification of rGFP

Experiment 8

Experimental Objectives

This week’s experiment will center on the multi-column purification of two pooled elution fractions of rGFP from the Ni⁺² agarose affinity column (Exp 4) through an anion exchange column and a gel filtration column. Various fractions from these columns will be analyzed for activity (Exp 4 protocols), total amount of protein (Exp 5 protocols), and rGFP purity using SDS-PAGE (Exp 6 protocols).  

Column 1 – Ni²Agarose Chromatography

This column procedure was described and performed in Experiment 4.  Assume you still have a total of 500ul each of the W1-W6 and E1-E6 fractions that you stored in the freezer.

Column 2 – Anion Exchange chromatography

1. Preparing your sample. Thaw and briefly vortex the two elution fractions from column 1 that had the highest rGFP activity.  Remove 400ul of each these fractions and mix together in a new centrifuge tube (freeze the remaining 100ul for analysis in Exp 5-7). This procedure is typically called “pooling samples.” Add 200ul of 100mM Tris (pH8.8), 10mM NaCl buffer to the pooled sample so that you have a total volume of 1ml to apply to the DEAE-52 column below. 

• Prepare a 5ml bed volume DEAE-52 resin syringe column using a 50% slurry of DEAE-52 pre-equilibrated in a 100mM Tris (pH8.8), 10mM NaCl buffer.  Use the same type of equipment and principles learned in Exp 4 to make this column.  Adjust the flow rate of the column so you are collecting about 8 drops per minute (about 250ul/min).

• Load your 1ml pooled sample (step 1 above) onto the column and begin collecting 1ml fractions. Label the first 1ml of eluate as F1.

• After collecting fraction F1, begin applying 9ml of wash buffer to the column and collect 1ml fractions. Label these fractions F2-F10.

• After collecting fraction F10, begin applying 10ml of a 100mM Tris (pH8.8), 50mM NaCl buffer to the column and collect 1ml fractions. Label these fractions F11-F20.

• After collecting fraction F20, begin applying 10ml of a 100mM Tris (pH8.8), 150mM NaCl buffer to the column and collect 1ml fractions. Label these fractions F21-F30.

• After collecting fraction F30, begin applying 10ml of a 100mM Tris (pH8.8), 200mM NaCl buffer to the column and collect 1ml fractions. Label these fractions F31-F40.

• Save fractions F1-F40 and analyze for activity, total amount of protein, and purity (data in lab report):

1. Determine the fluorescence present in 200ul of each fraction using the equipment and procedure from Exp 4 (return this 200ul back to the appropriate F1-F40 tube).
2. Determine the total amount of protein present in each fraction using a 50ul Bradford assay procedure explained in Exp 5. Remember, once a protein sample is used in the Bradford procedure, it is no longer usable.
3. Determine the percent purity of rGFP of the most active fraction using a 50ul sample for a SDS-PAGE procedure explained in Exp 6. Remember, once a protein sample is used in the SDS-PAGE procedure, it is no longer usable.

Column 3 – Gel Filtration (size exclusion) chromatography

• Preparing your sample. Pool the three fractions from column 2 that had the highest rGFP activity. (Remember, you used some of the sample in the steps above for Bradford/SDS-PAGE analysis.) Add 300ul of 100mM Tris (pH8.8), 10mM NaCl buffer to the pooled fractions so you have total volume of 3ml to apply to the gel filtration column below. 

1. Prepare a 25ml bed volume Sephadex G-100 syringe column using a 25% slurry of Sephadex G-100 pre-equilibrated in a 100mM Tris (pH8.8), 10mM NaCl buffer.  Use the same type of equipment and principles learned in Exp 4 to make this column.  Do NOT let this column run dry.  There must always be buffer on the top.  Adjust the flow rate of the column so you are collecting about 16 drops per minute (about 500ul/min).

1. Load your 3ml pooled sample onto the column and begin collecting 1ml fractions. Label the first three fractions GF1, GF2, and GF3.

1. Wash the column with 97ml of 100mM Tris (pH8.8), 10mM NaCl buffer.  Continue collecting 1ml fractions. Label them GF4 through GF100.

1. Analyze GF1-GF100 for activity, total amount of protein, and purity (data in lab report):

1. Determine the fluorescence present in 200ul of each fraction using the equipment and procedure from Exp 4.  See the Data Set
2. Determine the total amount of protein present in each fraction using a 50ul Bradford assay procedure explained in Exp 5. Remember, once a protein sample is used in the Bradford procedure, it is no longer usable.
3. Determine the percent purity of rGFP of the most active fraction using a 50ul sample for a SDS-PAGE procedure explained in Exp 6. Remember, once a protein sample is used in the SDS-PAGE procedure, it is no longer usable.

1. Pool the four GF fractions (total final volume 3.6ml since you used some of the sample in the steps above for Bradford/SDS-PAGE analysis) that contained the highest rGFP activity.  Store in the freezer for future use.

BIOL 3380

Experiment 8 – Lab Report

Multi-column purification of rGFP

Name:

Lab section:

Instructor:

Graduate TA:

Assigned data set:

Date:

Experiment 8 – Lab Report

Multi column Purification of rGFP

To save time, simply edit this word document by inserting your answers after each question/space. Please denote your answers by using bold, italics, or colored font. Do NOT renumber/reorganize the questions.

“Showing your work/calculations” can be achieved by typing it out, pasting in a clear image of your handwritten work, or using handwritten annotation on the document (if you have the technology to do so).

Insert graphs (handwritten on graph paper or electronically constructed with horizontal/vertical gridlines) as a clear image with appropriate titles, labeled/scaled axes, and standard deviation bars when appropriate.

• My assigned data set for experiment 4 was:________

(1pt) Which two fractions did you pool from column 1 (Ni⁺² agarose) to apply to column 2 (anion exchanger)?

• My assigned data set for experiment 4 was:________

(3pts) What was the amount of rGFP activity that you loaded onto column 2? Show your calculations for credit.

• (3pts) Based upon the protocol, if the highest fluorescence was found in fraction F32, does rGFP carry a net positive, net zero, or net negative charge? Explain your reasoning for full credit.

Use the elution profile above for the Sephadex G-100 Column to answer the following:

• (3pts) Based upon the protocol, predict a reasonable void volume for the Sephadex G-100 column? Explain your reasoning for full credit.

• (3pts) Which peak represents the largest protein/protein complex?  Explain your reasoning.

1. (2pts) What could you do in future purification procedures to sharpen peak 2 like peak 1? Explain your reasoning.

Step	Sample	Total protein (ug) Based on Bradford Assay	Activity (RFU) based on fluorescence	% purity based on SDS-PAGE
A	GCE (1ml)	450	48,000	5
B	Pooled E fractions from Ni+2 Agarose less	98	35,000	40
C	Pooled F fractions from anion exchanger	9	30,000	85
D	Pooled GF fractions from Sephadex G-100	5	29,000	90

Using the Exp 8 protocol and the purification table above to answer the following questions:

• (2pts) How much rGFP in ug was present at the beginning of the purification process? Show your calculations for credit.

• (2pts) Going from what two steps resulted in the least loss of activity?

• (2pts) Going from what two steps resulted in the largest fold increase in purity?

• (2pts) Going from what two steps resulted in the greatest fold decrease in total protein? Show your work for credit.

• (2pts) What was the yield in ug of rGFP finally purified using this purification process? Show your calculations for credit.

• (2pts) Is specific activity a good indicator of increasing purity?  Yes/No.  Show your work for credit.

• (3pts) In an attempt to save money and time, which step would be the best step to remove without significantly altering the purity of rGFP? Justify your answer