Weeks 8 & 9: University of Iowa Department of Biochemistry Maria Spies’ Lab

July 15th, 2013

Scott Casey ’15 Dimensions Fellow in Research

My eighth week in Maria Spies’ lab was spent restarting a cell line of HEK293T in order to transfect it to create FANCJ; hopefully with a higher concentration this time.  The cell line was successfully restarted on Tuesday by switching to a different set of HEK293T stocks and was scaled up over the weekend in order to have enough cells to create FANCJ.  Once the cell line had been scaled up to 20 plates the transfection was carried out, cells collected, and then the protein was purified out using the same crude purification process as the last two times.  The purification was then prepped for a heparin column chromatography.  This is similar to the gel column chromatography that was done with prep 2.  The only difference is that the column is filled with Heparin instead of gel.  I am used to dealing with Heparin in a medical role where it is used as an anticoagulation treatment for strokes, Atrial Fibrillation, and other conditions.  Heparin is useful in this instance because it is a polarized molecule and thus mimics DNA’s binding characteristics which mean that something that binds to DNA is also likely to bind to Heparin.  The protein is then eluted by gradually increasing the salt concentration within the column which forces the protein to unbind.  The protein and the flag peptide unbind at different salt concentrations which allows us to separate the protein from the flag peptide.  The Heparin column will be run next week.

These two weeks have been a little stressful because of the need to restart the cell line and the knowledge that Maria’s next grant application is dependent upon having some results to show from these experiments on FANCJ.  Knowing how important this set of experiments is and that I only have a month and a half in the lab makes this feel a lot like the school year on the block plan.  It feels like home.

Week 7: University of Iowa Department of Biochemistry Maria Spies’ Lab

July 3rd, 2013

Scott Casey ’15 Dimensions Fellow in Research

The fractions most likely to contain FANCJ were run on an SDS-Page gel which showed a very faint signal in Fraction 7 for FANCJ.  Fraction 7 was analyzed on the Nanodrop spectrophotometer at 280 nm wavelength but there was no detectable concentration of FANCJ.  Because the concentration of FANCJ in the fraction was so low as to be unusable for the planned experiments, a new cell line of HEK293T was started from frozen stock.

This week was both stimulating and frustrating.  Frustrating because my preparation was not ready for experimentation and stimulating because Masa and I had to work on figuring out a way to increase the concentration of FANCJ produced by the transfection.  It’s always amazing to think about the differences between lab at the University of Iowa and at Cornell.  Labs at Cornell are great, but what happens is what happens and what you write up.  With more time to research, there is time to try procedures again, modify them and optimize their results.

Week 6: University of Iowa Department of Biochemistry Maria Spies’ Lab

June 25th, 2013

Scott Casey ’15 Dimensions Fellow in Research

A Western Blot was run on the eluate from the crude filtration and several fractions from the gel chromatography; unfortunately it showed that while the Eluate from the crude filtration did indeed have FANCJ in it, none of the fractions did.  A second Western blot was run on the remaining fractions to determine if any of them contained FANCJ.  The two Western Blots together showed that none of the fractions contained FANCJ.  It was then determined that a SDS-Page gel should be run on the fractions most likely to contain FANCJ based on typical elution volumes for proteins of a similar size (in kDa).

Will Jones, one of the research assistants in the lab was very helpful in figuring out how to confirm the presence or absence of FANCJ in my samples.  It is great to be working in a lab where everyone is willing to help with a problem and find the solution.

Week 5: University of Iowa Department of Biochemistry Maria Spies’ Lab

June 18th, 2013

Scott Casey ’15 Dimensions Fellow in Research

The purified plasmid containing the FLAG-tagged FANCJ was run on an agarose gel to confirm the proper vector and insert were present. The Gel showed that they were.

Agarose Gel of 6 samples of pcDNA3 containing FLAG-tagged FANCJ

Agarose Gel of 6 samples of pcDNA3 containing FLAG-tagged FANCJ

Following the confirmation of FANCJ in the purified plasmid, the cultures of mammalian cells, HEK293T, were transfected and left to incubate overnight.  The protein from the transfected cells was purified the same as before but with an extra gel chromatography purification step after the crude filtration.  Some of the eluate from the crude filtration was saved to be analyzed for FANCJ content along with the fractions from the gel chromatography.

Week 4: University of Iowa Department of Biochemistry Maria Spies’ Lab

June 11th, 2013

Scott Casey ’15 Dimensions Fellow in Research

A bulk fluorometry was run on the elution from the crude filtration of last week to determine the elution’s concentration of FANCJ.  This assay is possible because of the structure of FANCJ which contains an iron-sulfur cluster.  This cluster quenches the fluorescence of cytochrome 3 and 5 when in close proximity, such as when it binds to DNA tagged with cytrochrome 5.  The DNA construct used for this assay is forked DNA that has a cytochrome 5 tag on one of the single strands of DNA that make up the fork.  When FANCJ binds to the single strand it quenches the fluorescent signal from the tag which can be detected by the fluorometer.  When the quenching first levels off the FANCJ and DNA are present in the solution at a 1:1 ratio which can then be used to back calculate the concentration of the elution.  The downside to this assay is that it requires a somewhat large amount of protein in a relatively high concentration in order to be effective which is where I ran into problems because my elution was not concentrated enough.  The bulk assay showed no noticeable change in the fluorescent signal of the DNA throughout the titration.  Because of the low concentration of FANCJ in the elution a second round of transfections was begun from the cell line that I had been continuing since the last transfection.  The first transfection had used the last of the lab’s stock of the pcDNA3 plasmid that contained the code for the flag tagged FANCJ.  The first step in this second round of transfections was to inoculate cultures of E. coli containing the plasmid and then purify the plasmid to make more stocks.

Working with the E. Coli was a very familiar experience since I spent most of last summer working in a lab that was attempting to create a process for manufacturing BDO in E. coli; unfortunately the first round of E. coli cultures did not grow properly and so I had to reinoculate.

The setbacks this week were frustrating but I still find research to be interesting and enjoyable.  The setbacks are just new problems to be tackled and solved.

Week 3: University of Iowa Department of Biochemistry Maria Spies’ Lab

June 4th, 2013

Scott Casey ’15 Dimensions Fellow in Research

The frozen cells from the previous week were thawed and put through a process called crude filtration.  The cells were lysed and the lysate was then filtered before being mixed with beads coated in an anti-FLAG protein that will bind to the flag tag on our FANCJ and hold it to the beads.  The bead and lysate mixture was then incubated for 24 hours on an automated rotator.  The FANCJ was then washed off of the beads and the elution was run on a SDS-PAGE gel along with samples of what flowed through the beads (what didn’t bind) and a positive FANCJ control.  The gel was then processed using the Western Blot protocol and imaged.  The Western Blot showed that the eluate did contain FANCJ and that the Flowthrough didn’t contain a noticeable amount.  The next step was to run a bulk fluorometric assay on the eluate to determine its concentration next week.

Western Blot from Week 3

Western Blot from Week 3

Each sample was run twice on this gel.  The right side of the gel had overflow issues and so was disregarded as all samples on that side were contaminated.  The left side shows the positive control at around 150 kilodaltons and the eluate at a similar size.

The Western Blot was very similar to the Southern blot that I had done in Cell and Molecular Biology.  The tags that were used were different but the overall process was very similar.

Week 2: University of Iowa Department of Biochemistry Maria Spies’ Lab

May 28th, 2013

Scott Casey ’15, Dimensions Fellow in Research

The first week of my internship at Maria Spies single molecule biochemistry lab was spent familiarizing myself with the lab and general protocols and beginning the work of creating the protein stocks that I need for my investigation of FANCJ and it’s binding and motility properties.  This first week was both very busy and very slow.  Without stocks I was unable to do much.  In order to create our protein we transfect mammalian cells with the DNA for the protein including a FLAG tag that can be used to purify it out later.  The first step in this process is to grow a culture of mammalian cells for transfection which is a week to a week and a half process.  On the other hand I had several research papers and lab papers to read and understand as well.

Everyone in the lab is very helpful with finding protocols and supplies, although I think that sometimes they must get frustrated with it since there are two summer interns in the lab and two undergraduate interns from the University of Iowa which makes for a lot of questions, but also provides a lot of extra hands for getting experiments done. When I started at the lab I was surprised to find out that both Maria Spies and my immediate mentor, Masayoshi (Masa) Honda, spoke Japanese which I started studying at the end of the school year.

The second week started out with a slight setback.  The cultures of mammalian cells that I had planned to transfect on Tuesday had not grown sufficiently for the transfection to be effective.  The transfection was carried out successfully on Wednesday though.  The cells were incubated for 24 hours after the transfection to allow the protein to fully express.  On Thursday I collected the transfected cells and froze them for storage in preparation for extracting and purifying the protein on Monday of the next week.

This week didn’t seem like it had much work in it looking at the hours spent in the lab, but most of my time was spent making sure that I understood the questions of our experiment; this is a goal that I am continually pursuing.  It is amazing how long research takes.  At the end of the second week, I had barely even started on the production of my protein stocks, let alone started my experiment.  It might be a good thing that I am planning on spending nearly 4 months here.

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