Protein Isolation and Analysis- 2013


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The objective of this experiment is to isolate total protein from your plant tissue. Next week we will run equal amounts of total protein from each of your plants on an an acrylamide gel and Western Blot it in order to quantify how much Rubisco Large Subunit is present in this total protein mix.

It is actually very easy to isolate proteins and requires very few steps compared to isolation of DNA or RNA. However, you do need to be careful when isolating proteins as the tissue and proteins need to be kept cold to prevent them from degrading. We will also use some proteases inhibitors to prevent protein degradation over the next week until we can run our Western Blot. In reading through this protocol please make sure you understand what each ingredient is in the buffer we are using and why it is important.

After we have isolated our proteins today, we will determine how much protein we will run on the gel next week. You will use the DC protein assay (BioRad) to determine protein concentration. This assay is based on a technique called the Lowery Assay. It requires two steps and about 40 min to perform. In the first step, a reaction occurs in which aromatic assays serve as a catalyst to convert Cu +2 to Cu+1. The second step uses Cu+1 and the aromatic amino acids, tyrosine and tryptophan, to reduce the Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate) to a blue colored compound. We will be performing this reaction on our proteins and on standards of known protein concentration. From a curve of the absorbances of the standards read at 750 nm to detect how much Folin-Ciocalteu reagent is reduced, the concentration of your proteins can be determined. An absorbance of 750 nm is used in this experiment because plants have few light pigments that interfere with light absorption at that wavelength. Make sure as you read through protocols that you understand how to do all of the calculations for these experiments before coming to class as that will help you to make sure you can most effectively use your class time to perform the experiments.

Overall, read through the protocols before you get class keeping in mind the following:

1. What is the basic procedure? What am I trying to accomplish overall?

2. Why am I doing each step? What is the result of the step?

3.Do I understand the calculations that will be required of me in class?


PROCEDURE:

1. Obtain ice, and place in it QB buffer {which contains 100mM potassium phosphate buffer pH= 7.8 (buffer which keeps proteases from functioning well as their ideal pH is fairly low), 1mM EDTA (chelates or binds up Mg2+ required by some proteases for activity), 1% Triton-X-100 (a detergent which helps to break protein membranes), and 10% glycerol (a thickener which will help later when we freeze the protein)}, 2 mortar and pestles from the refrigerator and 1 vial of protease inhibitors (special chemicals designed to specifically inhibit certain plant proteases that are extremely prevalent) in it. Clearly label two 1.5 ml microcentrifuge tubes for each plant tissue from which you want to obtain protein with the name of the plant and your groups initials.

2. Add the100 ul vial of protease to the 10 ml of QB buffer and store this on ice.

3. From each plant cut up 2g of fresh plant tissue as fine as you can with a razor blade. Place this plant in a baggie and store in the -80 freezer. It will be used later in the course for isolating DNA.

4. From each plant cut up 1 g of fresh plant tissue as fine as you can with a razor blade. Place this into a mortar with approximately 1 ml of cold QB buffer containing protease inhibitor and a pinch of clean sand. Grind until smooth with a pestle. It is very important to grind the tissue well. You may need to add more buffer but the final product should be about the concistency of slightly watery toothpaste. Keep the mortar and pestle and plant tissue as cold as possible during this process.

5. Transfer 1 ml of slurry into a 1.5 ml microfuge tube using a rubber policeman or a clean gloved hand. Place the tube on ice and peform the steps 3 and 4 with each plant tissue. You will have to wash out the motar and pestles between plants as we only have two per group.

6. Spin your samples at top speed in a microfuge at 4 degrees C for 15 minutes. Remember to make sure that all centrifuges and microfuges are balanced and do not walk away from any centrifuge or microfuge until it is up to full speed.

7. Transfer the liquid supernatant into a second (new) microfuge tube.

8. Sometimes excess tissue is transferred over into the second microfuge tube. If this is the case, spin a second time for about 10 minutes and transfer this supernatant into a third microfuge tube.

9. Store the samples in ice only until you have finished your DC protein Assay, then freeze the samples in liquid nitrogen and put them in the -80 degree freezer until we are ready to use them on a Western Blot.

10.Set up for the DC Assay. You can do this while your samples are being spun down. This assay will show a color change dependent on the amount of protein present in the sample. The assay is time and temperature dependent and thus the samples need to be run right along side of the standards and you do need to keep some thought to how you time the entering of each ingrediant into the assay for best results.

A. First, you will want to determine how many samples you need and obtain and label a cuvette for each of these samples. Here is a list of the cuvettes you will need to set up.

          1) 0 mg/ml Standard (QB Buffer with no protein). This will be used to blank the machine.
 
           2) 0.25 mg/ml standard
           3)  0.5 mg/ml standard
           4)  0.75 mg/ml standard
           5)  1.0 mg/ml standard
           6)  1.25 mg/ml standard


           7) Your protein sample diluted 1:20 done in duplicate
           8) Your protein sample diluted 1:200 done in duplicate

(Note: We want your plant samples to have an absorbance reading above 0.25 mg/ml and below 1.25 mg/ml so that they fall on the curve. If it is too low or too high it won't be very accurate. Unfortunately, from past experience we really see a wide variety of concentrations so it is hard to predict how to have you dilute it to make sure you get a sample that will have a reading in this range. We especially have problems with numbers being too high. Thus, for your three samples, I recommend that you take your protein and dilute it by 1: 20 and 1:200. If you have a plant that was really watery or took a lot of QB buffer you may consider also doing a 1:4 dilution in duplicate in addition to those above to make sure you have something that falls on the curve. You will need 100 ul of diluted protein in each assay. Our pippettes in lab will accurately pippette only down to 2 ul as the lowest volume so think about how you will make these dilutions, you will need to do a seriel dilution in order to obtain a 1:200)

B. Make Reagent SA (Reagent A is the The Copper-containing reagent used in the first color change reaction, Reagent S needs to be mixed with it so that the detergent in our QB buffer can't interact with reagent A). The SA reagent should be stored at room temperature after it is made.

           1)   Count the number of tubes you will need from step A enter that number here_____________

     2)   Determine the amount of Reagent A you will need. You will need 500 ul of Reagent A for every cuvette.

     This means you need # from step 1 X 0.5 ml = ______________ ml of A.

           3)  Determine the amount of Reagent S you will need. You will need 20 ul of reagent S for every ml of Reagent A that you need.

           This means you need # from step 2 X 20 ul= ________________ul of S.

           4)  Place the appropriate amounts of S and A into a 15 ml tube and vortex.
           
 
C. Pipet 100 ul of standard (make sure you vortex and spind down all standards before using them) or sample diluted as appropriate into each labeled cuvette.
 
D. Add 500 ul of reagent SA and vortex the tubes. This is when reaction 1 occurs so make sure each sample is exposed to SA for an equivalent amount of time before adding reagent B.
 
E. Add 4.0 ml of reagent B into each test tube, cover with parafilm and vortex immediately. Reaction 2 will now start to occur.
 
F. Incubate samples for 15 minutes. During this time set up a computer to enter your absorbances and make your standard curve and clean up your area as much as possible.
 
G. Read Absorbances at 750 nm. First read the sample with and use it to zero the machine. The absorbances will be stable for about one hour.
 
H. Create a scatter plot on the computer using excel of your standards including the blank (should be 0 mg/ml) (on Y axis) vs. the standards absorbance at 750 nm (on X axis). Place a linear trend line on the graph and obtain an equation for the line and an R2 value. If the R2 value is less than 0.95 consult instructor.
 
I. Examine all six of your sample absorbances form the 1:2, 1:20 and 1:200 dilution and determine which ones have an absorbance higher than the 0.25 mg/ml sample and lower than the 1.25 mg/ml sample. Average these absorbances and plug them into the equation to determine the concentrations of your proteins. Once you have plugged them into the equation don't forget to account for the dilution factor. If none of your samples fall on this curve you will need to repeat the procedure again with a new standard curve and an adjusted sample volume.
 
J. When running your Western Blot you will need to load as much protein as you can into the wells next week with a maximum of 300 ug due to the likely-hood of the protein degrading between now and next week. So what you need to do is take your lowest sample and figure out how many ug of protein you can run from that sample in 30 ul. Then use that number of ug to determine how many ul of protein you will need to run from your other samples. If the amount of protein you can load from your lowest sample is less than 10 ug please see Dr. Rife.
Make sure you save this graph as you will want it for your lab report and record your protein concentrations below or in a place that you will remember to bring to class next week! Before leaving for the week you must have this chart initialized by your instructor.
Name of Protein Sample
Concentration of Protein Sample (ug/ul)
ul of Protein Needed to Load ______ ug on a Western Blot (see step I)
ul of QB needed to bring final volume to 30 ul
1.      
2.      
3.      
4.      
5.      
 
 

TKR 8/15/13