protein isolation from living cells

Question 1

part a: isolation of protein from living human cells

Answer 1

1) pour the culture medium (liquid) from the tissue culture flask into the waste beaker containing virkon (pink disinfectant)
2) add 2ml of phosphate-buffered saline (PBS) to your flask : gently swirl the solution over the surface of the cells, then remove and discard medium as before
3) using a clean pipette , add 1ml of fresh PBS to the flask
4) scrape the cells into the PBS using the cell scraper. Be sure to scrape the whole of the surface of the flask to remove the attached cells. The PBS will start to become cloudy as the cells are scrapped of the flask and into the liquid
5) using a 1ml pipette , transfer the PBS/cells into a clean micro centrifuge tube. label the top of your tube with your group number.
6) centrifuge the tube at 800g for 3minutes
7) remove the supernatant (liquid) and discard - your cells/protein are in the pellet
8) add 20 ul lysis buffer to the pellet and pipette up and down slowly , keeping the tip in the liquid , to re suspend the cells in the microcentrifuge tube.
9) close the micro centrifuge tube tightly and place on ice for 10 minutes , vortexing after 5 minutes.
10) the cells have now broken open and released their proteins into the solution . recentrifgue the tube at 3000 rpm for 5 minutes.
11) the proteins are now in the supernatant (liquid) and cellular debris is in the pellet - remove the supernatant carefully and place it in to a clean microcentrifuge . this is the isolated protein sample that you will use for the rest of the experiment

Question 2

Part B - estimate protein content of isolate using the BCA assay

Answer 2

The BCA protein assay is a detergent compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. This method combines the well know reduction of cu2+ to cu1+ by protein in an alkaline medium (the buret reaction) with the highly sensitive and selective colour metric detection of the cuprous cation (cu1+) , using a unique reagent containing bicinchoninic acid. The purple -coloured reaction product of this assay is formed by the chelation of two molecules of BCA with one cuprous ion. this water soluble complex exhibits a strong absorbance at 562 nm that is nearly linear with increasing protein concentrations over a broad working range (20-2,000 ug/ml)

Question 3

preparation of protein standards

Answer 3

you have been provided with 1.5 ml of a 2mg/ml bovine serum albumin (BSA) stock solution to make up a series of standard protein solutions
before you start , label 9 microcentrifuge tubes from A to I

Question 4

BCA WORKING REAGENT

Answer 4

you have been provided with 3ml working reagent (WR) in a labelled tube . this is the clear green solution

Question 5

Part B assay protocol

Answer 5

pipette 25 ul of each standard and your two isolated protein samples (neat and diluted samples) into different wells of a micro titre plate. make sure that you know what you have put in each well.
add 200 ul of the WR to each well and mix well by gently shaking the plate
cover and incubate the plate at 37 degrees for 30 minutes in an incubator
measure the absorbance of all the samples at 562 nm
subtract the absorbance measurement of the blank (sample I) from the absorbance measurement of all other samples
prepare a standard curve by plotting the corrected absorbance value for each BSA standard versus its concentration in ug/ml . use the standard curve (equation of the line) to determine the protein concentration of your isolated protein samples. ( y=mx+c)

Question 6

Part c - separate isolated proteins using the SDS-PAGE page technique

Answer 6

sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) is a technique commonly used in research laboratories to detect proteins of interest in for example cell supernatants . proteins in a sample can be separated by applying an electrical current to the protein sample which has been loaded to polyacrylamide gel. the cell is not solid but contains pores of differing sizes which produce a sieve-like effect on the proteins as they move down the gel towards the anode. with larger proteins being retained at the top of the gel and smaller proteins migrating further down the gel. prior to electrophoresis , sample treatment buffer containing -mercaptoethanol and SDS is added to the protein sample and the proteins denatured by boiling to disrupt any secondary , tertiary or quaternary structure and produce a linear structure . The addition of SDS also coats the protein in detergent giving it an overall negative charge. the proteins in the sample will therefore migrate solely according to their molecular weight and not according to either their charge or their shape .

Question 7

Part C assay protocol

Answer 7

1. Take 25 l of your isolated protein sample from Part A and place in a new microcentrifuge tube.
2. Add an equal volume (25 l) of sample treatment buffer to the tube and mix. DO THIS STEP IN A FUMECUPBOARD BECAUSE THE BUFFER HAS A VERY STRONG SMELL.
3. Close the tube tightly, stick the lid down with sticky tape, label with your group number, and boil for 10 min.
4. Once the sample has cooled slightly, give it to the demonstrator to be loaded on to the gel.
5. Once all the groups have had their samples loaded onto the gel, an aliquot of prestained standards will be added to an outside well. This solution contains a number of coloured proteins of known molecular weight which can be used for approximating the molecular weight of unknown proteins in your sample.
6. The gel will be run at a constant voltage of 200 V for approximately 45 min or until the proteins have reached the bottom of the gel.
7. Once electrophoresis is complete, the gel cassette will be opened carefully and placed in a container with Coomassie Blue stain for 1 h. This solution stains proteins blue and after staining, excess colour is removed from the gel by washing it in water for 30 min (a process called destaining). The proteins will retain the blue colour and you will be able to see them.
8. A photo of the gel containing your sample will be put on blackboard for you.
9. From this photo you will be able to approximate the molecular weights of the most abundant proteins in your sample.