Protein Isolation and Analysis

Back to Lab Schedule

It is actually very easy to isolate proteins and requires very few steps compared to isolation of DNA or RNA. You do need to be careful when isolating proteins as the tissue and proteins need to be kept cold to prevent them from degrading. Think about the ingrediants of the protein isolation buffer QB, you should have some idea of what each of the ingrediants in the protein isolation buffer does.

To determine how much protein we have we will use for Western Blotting, you will use the DC protein assay (BioRad) based on the Lowery Assay to determine protein concentration. This assay requires two steps and about 40 min to perform. In the first step, a reaction occurs in which Cu +2 is reduced 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.  This reaction is thus detectable by measuring in the range of 500 to 750 nm. We are going to use 750 nm to detect how much Folin-Ciocalteu reagent is reduced. Why would 660 nm, the usual wavelength that this assy is performed at not be appropriate for plants? Can you think of other possible limitations of this assay?

In order to figure out how much protein you specifically have each group will need to make up a standard curve of the protein concentratoin of known samples vs. the their absorbance at 750 nm at the same time you make up your samples because this reaction is temperature and time dependent. This curve will be used to convert the absorbances obtained from your samples into protein concentrations. Both your samples and the samples in the curve need to be assayed at the same time and with percision as the samples are light sensitive so a longer exposure to light can make a difference in the reading.

1. To isolate your protein place 1g of fresh plant tissue that has been finely cut with a razor blade into a mortar with approximately 2 ml of cold extraction buffer and a pinch of clean sand. (We are using QB buffer which contains 100mM potassium phosphate buffer pH= 7.8, 1mM EDTA, 1% Triton-X-100, and 10% glycerol). 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 concistancy of slightly watery toothpaste. Keep the morter and pestle and plant tissue as cold as possible during this process.

2. Transfer 1 ml of slurry into a 1.5 ml microfuge tube using a rubber policeman. Place the tube on ice until you have all of your samples ready.

3. Rinse mortar and pestle (and any other paraphernalia that came into contact with the sample)to remove all traces of sample, make sure plant tissue to be used next time is kept cold (place in bag in -80) and proceed to the protein isolation of the next plant sample.

4. Spin your samples at top speed in a microfuge at 4 degrees C for 15 minutes.

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

6. 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.

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

8.Set up for the DC Assay. You can do this why your samples are being spun down. To do this assay you must set up tubes with a series of standards along with three tubes of your samples. You will compare your samples to the standard tubes to determine your samples concentration. The samples and standards must be done at the same time to prevent differences in timing and temperature from being a factor in determining the amount of protein in your samples.

A. First determine how many samples you need and obtain and label a cuvette for each of these. Eventually into each Cuvette you will put 100 ul of protein that has been dissolved in QB buffer to start your reaction. Here is a list of the cuvettes you will need to set up.
 
(For each of your plants: I recommend using three different dilutions of your protein to ensure that one of your samples abosorbances will fall within the values of the protein standards- one of your three should have 30ul of plant protein and 70 ul of QB, another 95 ul of QB and 5 ul of plant protein, a third 99 ul of QB and 1ul of plant protein)
B. Obtain, vortex, and centrifuge the protein standards to make sure they are well mixed. They have been prepared in the same buffer as your sample has been resuspended in.
 
C. Make Reagent SA (The Copper containing reagent). You will need 500 ul of SA for every cuvette labeled above. You need ________ ml of SA. To make Reagent SA you will want to mix 20 ul of reagent S for every 1 ml of Reagent A that you need. Reagent S helps to ensure that the detergent we are using will not react with and reduce the Copper. This means I will mix ________ ml of Reagent A (use the same amount as the amount of SA that you need) and __________ ul of Reagent S.
 
D. Pipet 100 ul of standards and samples into the tubes.
 
E. Add 500 ul of reagent SA and vortex the tubes. This is when reaction 1 occurs.
 
F. Add 4.0 ml of reagent B into each test tube and vortex immediately. Reaction 2 will now start to occur.
 
G. After 15 minutes, absorbances can be read at 750 nm. The absorbances will be stable for about one hour.
 
H. Create a standard curve on the computer using excel for each of your samples. If any of your samples do not fall on this curve you will need to repeat the procedure again with a new standard curve and an adjusted sample volume. Make sure you save this graph as you will want it for your lab report and make sure you understand how to calculate the amount of protein from the curve as this would be a good lab exam question.
 
I. Finally, to prepare for the future you will need to figure out and record in your notebook, how many ul of each sample you will need in order to load 30 ug of protein on an acrylamide gel. (The number of ul must be less than 30 ul to be able to fit on the wells of the gel along with the correct amount of loading dye so you may not be able to load 30 ug of sample per lane talk to me if this is the case.) Make certain you keep this data in a notebook you can locate it again easily - put a star by it ... make sure it will be clear to you in a few weeks what you are to do or you will have a late lab day in a few weeks. Keeping track of your data is very important as we are going to keep coming back to things for awhile in this class.
TKR 9/16/06