Virtual Drug Screening stream
Spring 2013
Lab: Protein characterization
Objective
The purpose of this lab is to use gel electrophoresis to analyze the samples you prepared in the
previous 2 labs: expression and purification labs. Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) will be used to separate proteins in your samples. Using the
electrophoresis and spectroscopy results you will estimate the molecular weight of the gbr22 protein
as well as the purity and yield of the final purified protein product. This data will then be correlated
to the UV-Vis spectroscopy measurements that were made in the last lab to estimate the
concentration of your protein solution.
Lab Safety
Many of the chemicals used in electrophoresis are toxic and can be absorbed through the skin. The
stains used to visualize proteins will stain skin and clothing as well. Always wear gloves while
handling gels and gel apparatus and be careful. Any equipment or supplies located in the gel
electrophoresis area should be assumed to be contaminated and treated with caution.
Background
When purifying proteins, there needs to be a method to monitor the success of each separation
step. While spectroscopy can be used to measure the concentration of proteins in solution, it can
not normally discriminate between different proteins in a mixture. The most common technique
used to follow expression and purification steps is sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). In gel electrophoresis, the samples are introduced at one end of a
porous gel and an electrical field is applied across the gel using a power supply.
SDS is an anionic detergent which denatures proteins and imparts a negative charge that is
proportional to its mass. This means that the distance of migration through the gel is related only to
the mass of the protein (since the charge/mass ratio stays the same for most proteins) and the
mass can be estimated by comparison to molecular weight standards.
Last lab you purified a purple protein, called gbr22, and collected samples from the different
purification steps. In this lab you will characterize the purified protein using gel electrophoresis.
Lab Timeline: Work in pairs
Day 1: Preparing Samples: 30min-1hr (this can be done in advance – i.e. another day)
Loading and Running gel: 1 hr
Staining gel: 1 hr
Destain gel: overnight (or 2)
Day 2: Take image of gel: 30 min
Dry gel: 2 hrs (can leave while drying and return at end)
Note: There is an overnight step for this lab. This can be extended to 2 overnights if necessary.
Discuss with the RE or mentor.
Also, if you have not done the Nanodrop® spectrophotometric quantification of your protein from
last lab – then do that this week as well.
Prelab calculations
1. Calculate the amount of 5x TGS buffer to use to make 500 ml of a solution that is 1x TGS.
2. Calculate the amount of 6x sample loading buffer to add to 50 l of a protein sample so that
the final solution is 1x.
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Spring 2013
Equipment and materials
Heat block – turn on to 95OC
Mini-PROTEAN electrophoresis tank and lid
Power supply and leads
TGS running buffer
Bio-Rad precast polyacrylamide gel
6x gel (or sample) loading buffer (don’t get the DNA loading dye by accident!)
Protein Samples #1-6 from Expression and Purification labs
Molecular Weight standards
Plastic container w/ lid
Imperial protein stain
Experimental procedure
Preparing the SDS-PAGE gel samples
Samples 1-6 were collected during the protein expression and protein purification labs. You will
need to obtain 6x gel loading buffer from the TA/mentor. The loading buffer contains glycerol, DTT
(a chemical that reduces disulfide bonds), SDS, and the dye bromophenol blue – this is different
than DNA loading dye – but both look the same (blue).
Sample 1 requires more preparation since it contains whole cells that were collected before
harvesting the culture. Prepare a counterbalance by pipetting 500 ul of water into a 1.7 ml
microcentrifuge tube. Using a benchtop microcentrifuge (refrigeration isn’t necessary), centrifuge
sample 1 for 5 min at 5,000 rpm. After centrifuging you want to retain the pellet. Remove the liquid
(spent media) and dispose of in the bleach waste container, resuspend the cell pellet in 200 l of
water by pipetting up and down until there are no more clumps, and add 40 l of loading buffer (you
may need to cut the end off of the pipette tip so that it will be wide enough to take up the very
viscous solution – also you can vortex the blue loading buffer).
Samples 2-6 are 50 l protein aliquots of the sequential purification steps. To each of these tubes
add the necessary amount of the 6x loading buffer (calculated in Prelab Calculation #2 above) and
pipette to mix. Place all the tubes containing samples 1-6 into a heat block at 95C for 5 min.
Remove tubes from the heat block and centrifuge the tubes for 2 min at 5,000 rpm.
Option: these samples can be stored in -4OC if you want to run the gel on another day
Assembling the electrophoresis module
A general diagram of the apparatus is shown to the right.
For detailed instructions, refer to the document ‘SDSPAGE using the mini-PROTEAN’ on Blackboard. You will
need: a mini-PROTEAN tank, cover with attached
electrical leads, gel clamping frame (electrode assembly),
buffer dam, 500 ml of 1x TGS buffer, prepared protein
samples, and molecular weight standards.
Obtain a precast gel from the TA/mentor and remove from
the packaging. Turn the plate upside down and pull off the
tape along the bottom to expose the slot of the gel (this
allows for the buffer and current to flow through the gel. If
it is not removed, your gel won’t run). This is necessary to
allow contact of the bottom of the gel with the outer buffer
reservoir. The gels come with a 10-well comb that is used
to form the wells. Remove the comb by gently sliding
straight up.
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Place the gel cassettes into the clamping frame with the shorter glass plates facing inward. If you
are running a single gel, use the buffer dam for the second gel position (NOTE: the buffer dams are
not disposable. Save these after your gel run). The gels and buffer dam should rest firmly against
the green gaskets. Slide the green arms of the clamping frame over the gels and lock into place.
Check with the TA/mentor to confirm that you have assembled the frame correctly.
Insert the assembly into the tank, making sure that the red
electrode jack matches the red marking on the inside of the tank. With the
buffer level markings on the front of the tank, the assembly will fit in the
position at the back of the tank. Fill the gel clamping assembly (upper
chamber) with 1x TGS buffer to the top edge and fill the tank (lower
chamber) to the level indicated on the front for 2 gels. You are now ready
to load your samples into the wells.
Sample loading and power conditions
Clear each lane by using a ~20 gauge needle and syringe. Take up a milliliter or two of TGS buffer
from the tank into the syringe and then inject into the well forcefully to clear it out. Clear out each
well in this same manner.
Usually, the first well is skipped to prevent gel ‘smiling’ – or distortion. So, into the second well, load
7 l of the MW standards (get from mentor) – write down the name, catalog # and company of the
MW standard that you used – what company is it from? And what Is the product number? You will
need it for the lab report. Use a 10 µl pipettor and let it fall gently into the well and sink to the
bottom.
If working solo:
empty----MW----1----2----3---4----5----6----empty----empty
If you are working as a pair, run the first persons samples (#1-6) then run the second person’s
samples (#4,5,6) on the right side of the gel.
MW----1----2----3---4----5----6----(4----5----6)
After the MW standards - you can load 20 l of each protein samples (#1,2,3,4,5,6) into each of the
remaining wells using a P20 micropipettor. Take care not to puncture the gel with the tips. Write
down which sample is associated with which well – so you don’t get confused.
NOTE: if you are having trouble seeing the wells – add a few grains of bromophenol blue
Place the lid on the tank, aligning the color coded plugs and jacks and insert the electrical leads into
the power supply. Set the timer on the power supply to 25 minutes to start out. Set the voltage to
200 V and select run. Verify that your gel is running by looking for rising gas bubbles. You stop
your gel when the blue line reaches a centimeter or two from the bottom of the gel. Do not let it go
too long or the low MW proteins may run off the bottom of the gel. If you let it run too short – you
won’t get good resolution on your gel.
Removing and staining the gel
After the gel electrophoresis is done, turn off the power supply and disconnect the leads. Remove
the tank lid and pour off and discard the running buffer. Remove the gel cassette by opening the
arms of the assembly and gently peal the tape and separate the two plates along the sides of the
cassette. Remove the gel by floating it off the plate into a large plastic dish of nanopure water. The
gel is thin and somewhat fragile so handle with care or it will tear. Rinse the tank, lid and assembly
with DI water and hang to dry. Using tape, label your dish with Name and Date.
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To remove excess SDS which will inhibit the staining process, wash the gel three times for 5
minutes each time with ~100 ml of nanopure water (‘washing’ means letting it sit in the dish while
shaking on the orbital shaker). Discard the water by pouring down the drain – but don’t lose your
gel! Mix the Imperial protein stain by inverting the bottle several times. Add a sufficient volume of
stain to completely cover the gel (~30 ml) and place on the orbital shaker for 1-1.5 hours until dark
bands are very visible. Label the Petri dish with your initials and the date. When finished with the
staining, pour the used reagent into the container marked for used stain – this stain can used again.
To destain the gel add rinse twice with Nanopure water then add enough nanopure water in the
Petri dish to cover the gel (~100 ml). Place a small folded tissue (KimWipe) in the dish (to soak up
excess stain) and wash the gel overnight on the orbital shaker to remove the background staining.
Next Day:
Take a preliminary picture of the gel incase it does not survive the drying process
Use a white piece of paper as the background and the digital camera. Make sure your bands are
distinguishable in the image.
Save a copy for yourself on webspace or a USB drive.
Post your image online to Wiki page.
Dry the gel
Materials: Whatman paper, saran wrap, gel,
labeling pen
(DO NOT write on the paper before drying – wait
til after!)
At one of the appointed times, take your gel
over to the Biotech lab to be dried so that you can save
it and put in your notebook. Cut a piece of Whatman
filter paper that is large enough to cover your gel plus
extra room for labeling. Wet the paper and put your gel
on top of this. Cover with Saran Wrap (or cellophane
– more permanent). Then lay them on the drying bed.
Lay the sealing gasket over everything.
Set temp to 75OC on Gradient cycle for 1.5 hrs.
Turn on vacuum and start cycle.
You can check your gel by lifting the lid - A dry gel should be warm over the entire surface. Once
dry, break the vacuum seal first by lifting the sealing gasket. Then turn off the vacuum pump. You
can trim around the paper that the gel is stuck to (but leave room for labeling). On the front of the
paper, Write Name, Date, VDS, and what the gel is – i.e. label what is in each lane.
Take a picture of your dried gel.
Flip a coin to determine who gets to paste the actual dried gel in their Lab Notebook. The other
person will need to print out a picture of it.
Questions that should be answered in the course of your Lab Notebook(estimating molecular
weight, purity and yield):
On the internet, look up the Molecular Weight standard that you used. You should find an image
similar to the one below. Print this out for your lab notebook and post an electronic copy for
your Wiki Lab Report.
Q: Now, estimate the MW of your purified protein using the standards as a reference.
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Virtual Drug Screening stream
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Examples of Bio-Rad Molecular Weight Standards
Q: How many other protein bands are present in the sample 5 lane? If there was one other
band of equal intensity, the estimated purity would be 50%.
Q: What do you estimate the purity of your final protein (sample 5) to be?
Lab Notebook:
Use hand drawn diagrams when applicable
Record the steps taken and include images of your gel
Online Report: use your page on the WikiSpaces (under Protein Labs)
Lab Report:
This report will encompass the last three (3) labs: protein expression, purification, and
characterization.
1,000 words total –
Since this is online – you must be efficient with your words! Make them count!
Title: think of something semi-creative
Intro words: this should have some background information on technique of protein
production and purification.
There is a good article here: Nat Methods. 2008 Feb;5(2):135-46. Protein production
and purification.
Also a good website: http://www.embl.de/pepcore/pepcore_services/index.html
End with an objective statement where you briefly sum-up what you set out to do in
this set of experiments. Include a brief hypothesis.
M&M 250 words: be succinct, concise, and brief while allowing an informed reader to be
able to replicate your work. Do not show calculations for prelabs.
Results: show images of your results that you have already taken
CAPTIONS: make sure you have good captions (captions don’t count against the
word limit)
Transformation plates images - Mention how many colonies you got. Flask
images, purple pellet images (include the weight of your pellet)
Images of Elution 1 and 2 in tubes, Nanodrop screen shots (of 280nm reading
for Elution 1 only) - Include yields from your Nanodrop spectrophotometry for your
protein amounts.
Show brief calculations for Beer’s law calculations. A=Ebc
Protein Gel images, Image of Ladder from web as reference (molecular weight
marker used – get from website of company)
Discusssion
Analyze your results, address sources of error, and answer any questions from the handout
Q: Why do we use lysozyme? Why do we use Benzonase/Cyanase?
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Q: Briefly, address how the HIS tag system works
Q: Explain what is in the sample after each step: Sample 1, 2, 3,4 ,5, and 6
Q: What is different about the Wash vs. the Elution buffer?
Q: State the size of your protein from the gel and compare it to the size you determined in
the protein purification lab
Q: address the purity of your sample
Conclusions
Recap what was done
State key finding(s)
Address future directions/applications of this work to VDS research
References (2-3)
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