Difference between revisions of "20.109(S22):M1D5"

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(Part 2: Concentrate TDP43-RRM12 protein elution)
(Part 2: Concentrate TDP43-RRM12 protein elution)
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#*Remove the cap from the filter unit, then pipette the TDP43-RRM12 protein solution into the filter unit and replace the cap.  
 
#*Remove the cap from the filter unit, then pipette the TDP43-RRM12 protein solution into the filter unit and replace the cap.  
 
#Centrifuge at 4500 g for 20 minutes.
 
#Centrifuge at 4500 g for 20 minutes.
#Transfer the liquid from the top of the centrifugal filter unit to a fresh microcentrifuge tube.
+
#Transfer the liquid '''from the top of the centrifugal filter unit''' to a fresh microcentrifuge tube.
 
#*This is the concentrated TDP43-RRM12!
 
#*This is the concentrated TDP43-RRM12!
  

Revision as of 18:04, 14 February 2022

20.109(S22): Laboratory Fundamentals of Biological Engineering

Sp17 20.109 M1D7 chemical structure features.png

Spring 2022 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       M1: Drug discovery        M2: Metabolic engineering        M3: Project design       


Introduction

Today you will perform the first of two functional assays aimed at assessing the small molecules that were identified using the SMM technology. In this assay, you will examine the effect of the small molecules you choose on aggregation. Protein aggregation refers to the biological process by which intrinsically disordered or mis-folded proteins associate to form an aggregate.

As discussed in lecture, the TDP43 protein contains an intrinsically disordered region that is prone to aggregation. TDP43 aggregates have been observed in both familial and sporadic ALS patients. For this experiment, we will examine the effect of the small molecules on the formation of TDP43 aggregates using the protein purified in the previous laboratory sessions.

Protocols

Part 1: Participate in Communication Lab workshop

Our communication instructor, Dr. Prerna Bhargava, will join us today for a discussion on writing figure titles and captions.

Part 2: Concentrate TDP43-RRM12 protein elution

To prepare TDP43-RRM12 for the aggregation assay, it is important to concentrate the protein that was prepared in the previous laboratory session. Concentrating the protein eliminates excess buffer and contaminants. To do this, a centrifugal filter with a 3 kDa cutoff will be used. The cutoff value refers to the size of the molecules that are able to pass through the filter -- molecules smaller than 3 kDa flowthrough the filter whereas molecules larger than 3 kDa are retained in the reservoir above the filter. One critical consideration when selecting a centrifugal filter is the molecular weight of the protein of interest. The protein should be larger than the cutoff value to ensure it is retained in the reservoir above the filter.

For timing reasons, the Instructors concentrated the protein that you will use in your experiment. The steps for the concentration process are included below for your notes.

  1. Retrieve the desalted "elution" sample of the purified TDP43-RRM12 protein.
  2. Add the elution to the centrifugal filter unit.
    • Remove the cap from the filter unit, then pipette the TDP43-RRM12 protein solution into the filter unit and replace the cap.
  3. Centrifuge at 4500 g for 20 minutes.
  4. Transfer the liquid from the top of the centrifugal filter unit to a fresh microcentrifuge tube.
    • This is the concentrated TDP43-RRM12!
Diagram of centrifugal filter and concentration protocol. (A) Centrifugal filter with 3 kDa cutoff is used to concentrate protein solution and remove contaminants. (B) Aliquots are collected at different steps of concentration procedure to assess protein expression and purity.

Part 3: Perform aggregation assay

Part 4: Draft Data summary slide for protein purity and concentration results

To get a headstart and further feedback from the Instructors, you will draft the slide that will present the TDP43 protein purification data for your Data summary today in class. With your partner, use the template below, the Instructor comments from your previous homework assignments, and the helpful hints from the Comm Lab workshop to craft a data slide with the required elements.

Individually you and your laboratory partner each crafted figures (with a title and caption) using the TDP43 protein purification results. In this exercise you will come together to decide how to best present these data for the Data summary!

Fa16 M1D7 data slide template format v2.png

Reagents list

  • centrifugal filter unit, 3 kDa cutoff (from Millipore)

Navigation links

Next day: Learn best practices for mammalian cell culture and seed CAD cells for TDP43-localization experiment

Previous day: Assess purity and concentration of purified TDP43 protein