protein isolation

Protein Isolation and Analysis- 2010

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. We will also have some proteases inhibitors in our proteins to prevent their degradation over the next few weeks as even in the freezer proteins can degrade over time. You should make sure you understand what each ingredient is in the buffer we are using and why it is important.

To determine how much protein we will use for Western Blotting on week five, 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. This reaction is thus detectable by measuring the abosorbance of the solution 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 assay is performed at, not be appropriate for plants? Can you think of other possible limitations of this assay?

In order to determine how much protein you have, each group will need to make up a standard curve by using protein solutions of known concentration. The absorbance change of the Lowery reagent versus the concentration of these solutions should have a linear relationship when graphed. Thus, this line can be used to determine the concentration of your isolated protein samples. This reaction is temperature- and time-dependent, so why would it be important to make up this curve at the same time that you assay your samples?

PROCEDURE (Make sure you read this completely before lab!)

0. To 10 ml of QB buffer which contains 100mM potassium phosphate buffer pH= 7.8, 1mM EDTA, 1% Triton-X-100, and 10% glycerol add 1 vial of protease inhibitors.

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. 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.

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 for DNA isolation next week is kept cold (place in bag in -80 freezer.) 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 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.

8.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 samples 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 determine how many samples you need and obtain and label a cuvette for each of these. Here is a list of the cuvettes you will need to set up.
1) A Blank – Use QB Buffer 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-?) Each of your samples x 3: (Note: We want your plant samples to have an absorbance reading above 0.125 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 in the following way: Sample 1: 1:2 dilution in QB, Sample 2: 1:20 dilution of your protein in QB, Sample 3: 1:200 dilution of your protein in QB. We will do each of these in duplicate. Note there is one thing to watch for- you must save 100 ul of your sample for the next lab so make sure you don't use it all. If your running low you might try a 1:4 dilution instead of a 1:2...)
B. Make Reagent SA (The Copper-containing reagent) needed for the color change assay. This reagent must be mixed right before use.
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 500 ul = _____ ul 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.
: G. Read Aborbances at 750 nm. First read the blank 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 one has an absorbance higher than the 0.25 mg/ml sample and lower than the 1.25 mg/ml sample. This is the absorbances you will want to plug into the equation to determine the concentrations of your proteins. If none 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 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.

I. Finally, 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 likelyhood 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.

TKR 9/16/06