Cellular Stress Response – Isolation

Background information: A typical eukaryotic cell contains tens of thousands of different , each involved in specific cellular processes. The heat-shock proteins were originally identified because they are produced in large quantities when cells are subject to heat-induced stress. Because subsequent studies have demonstrated that they are produced in response to a variety of stresses, these proteins are now often referred to as stress proteins. It is important to recognize that stress proteins have essential roles in non- stressed cells, where they function normally in the synthesis, transport and folding of non-heat-shock proteins.

The stress proteins are produced to protect the cell from situations that would lead to irreversible cell damage and ultimately to cell death. Under conditions of stress, the genes encoding the heat-shock proteins are activated and large amounts of the proteins are produced. When the stress is relieved, the level of these proteins returns to normal. These changes can be observed by examining the protein content of a cell using polyacrylamide (PAGE). In Drosophila, the most prevalent heat-shock proteins belong to the hsp70 family, with molecular weights of around 70,000 daltons. In today’s lab, we will begin a multi-week exercise in which we will assess the transcription of a specific gene that encodes for the HSP 70 protein.

The examination of the proteins in a cell requires several steps, including isolation, separation, and visualization. Isolation is accomplished by homogenizing a tissue sample in an appropriate buffer. Separation and visualization are accomplished by electrophoresis in polyacrylamide gels under denaturing conditions. Before loading the sample on the gel, the protein is denatured using a combination of a detergent, such as SDS; a reducing agent, such as mercaptoethanol; and heat. This ensures dissociation of the individual polypeptide subunits that are characteristic of many proteins.

Samples must be kept on ice as much of the time as possible during the homogenization process. This is because as cells are disrupted, their organellar contents are released into the homogenization solution. Organelles of note are peroxisomes and lysosomes. The contents of these organelles include proteolytic enzymes and other harmful substances that can quickly damage or digest the proteins in your extracted sample, thereby hindering your analysis.

After disrupting the tissues and cells of your Drosophila larvae, the total protein content of the samples will be determined using the Bradford protein and a standard curve. In this case, you will again be using bovine serum albumen (BSA) to produce a standard curve. In this week’s lab, however, your homogenized Drosophila larvae samples will serve as an “unknown” for which you must determine protein concentration.

Recall from the previous Bradford protein assay , it is possible to have too much protein in your sample such that the readings obtained are outside of the linear range of your standard curve. Because of this, you will need to dilute your extracted samples in different ways to ensure that at least one of your measurements is within the line that your standard concentrations produce—ideally within the middle of the linear range of your standard curve. Factoring in the dilution of your samples, you can then calculate the amount of total protein per unit of volume in your Drosophila larvae extraction samples.

Overall Objectives: Effectively extract and accurately quantify proteins from intact Drosophila tissues so that the level of transcription of a specific gene, that for HSP 70, can be analyzed in future weeks.

Experimental Procedure: 1. Obtain three Drosophila larvae samples: two experimental samples (37o and 42o) and another control (25o). There should be numerous larvae in each tube.

2. Label a clean 1.5ml microcentrifuge tube with each sample ID.

3. Keep all samples on ice unless you are manipulating them; return them to the ice as quickly as possible following manipulation.

4. Pipette 100 µl of ice-cold homogenization buffer into each tube.

5. Obtain one blue plastic homogenizer for each of your three samples and label three empty microcentrifuge tubes for your samples. Keep these tubes on ice for use in step 8.

6. Work with one of the sample tubes at a time, keeping the others on ice. Using a micropipettor, add 50 µl ice-cold homogenization buffer to the sample. Quickly homogenize the samples on ice until no tissue is visible. Use a new/separate homogenizer for each of your three samples.

7. When all three samples are sufficiently homogenized, centrifuge the homogenates at 10,000 xg (RCF) for 10 minutes to pellet any remaining debris to the bottom of the microcentrifuge tube.

8. Transfer as much of each homogenate as possible to the appropriate clean, labeled tube, taking care not to disturb the pelleted debris. These three samples will be your “unknowns” whose protein concentration must be determined. Keep the tubes on ice at all times.

9. Label two new microcentrifuge tubes, 1:2 and 1:10, for each of your three “unknown” Drosophila larvae homogenates. This should be a total of six tubes.

10. Make two dilutions of each of your three homogenates as follows:

• 1:2 –20 µl of your unknown sample + 20 µl of dH2O. Vortex briefly to mix. • 1:10 –20 µl of your unknown sample + 180 µl of dH2O. Vortex briefly to mix.

At this point, place your remaining protein samples in the freezer for use in the next lab.

11. Label two new microcentrifuge tubes, 1:2 and 1:10, for each of your three “unknown” Drosophila larvae homogenates. For each tube, add 40 µl of the appropriate dilution to 360 µl dH2O. NOTE: you should have six tubes total.

12. After completing the dilutions of your homogenates, acquire a tube of Bovine Serum Albumen (BSA) from your instructor. This tube contains BSA at a concentration of 100 µg/ml.

13. Prepare a 96-well plate by creating a “map” of each sample location. For the map, you will use each of the eight standard protein concentrations in the table below, as well as each each of the six homogenate dilutions you have prepared.

14. Label eight microcentrifuge tubes for the dilutions of BSA for the standard curve:

Know Concentration of BSA Volume of Volume of dH2O (mg/ml) and Tube Label Stock BSA 0 0 µl 800 µl 1 0.8 µl 799 µl 5 4 µl 796 µl 10 8 µl 792 µl 20 16 µl 784 µl 50 40 µl 760 µl 100 80 µl 720 µl 200 160 µl 640 µl

15. Add 200 µl of Bradford reagent to each tube (standards and homogenates) and vortex to mix. Incubate at room temperature for at least 5 minutes.

16. Transfer 250 µl of each of the diluted homogenate samples to each of two wells of a 96-well plate as indicated on your plate ‘map’

17. Use the plate reader to read the absorbance/OD at 595 nm.

Post-lab assignment: As with your previous laboratory, plot your standard curve as a line and provide the: a. Formula for the slope (y = mx + b). Remember that your R2 value should be 1 (or close to it) for a line; if it is not, discard any ‘outliers’ to generate the line (and equation) you will use to calculate the concentration of any unknown samples whose absorbance values are within the linear range of the standard curve. b. Calculate the protein concentration of your three unknown samples (two experimentals and one control) using the averages of any data points that are within the linear range of your standard curve. c. Next week, you will want to utilize 50 µg of protein per well of an SDS-PAGE assay. Given the calculated concentration of protein in your Drosophila homogenate, calculate the volume that will be used to generate that amount of total protein.