Virtual Drug Screening stream

Spring 2011
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. In this lab you will analyze the protein samples that you collected during
the 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 week 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 and UV-Vis spectroscopy.
Lab Timeline:
Day 1: Preparing Samples: 30min-1hr
Loading and Running gel: 1 hr
Staining gel: 1 hr
Destain gel: overnight
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.
necessary. Discuss with the RE.

This can be extended to 2 overnight if

1

and the dye bromophenol blue. 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. Calculate the amount of 6x sample loading buffer to add to 50 l of a protein sample so that the final solution is 1x. For detailed instructions. resuspend the cell pellet in 200 l of water by pipetting up and down until there are no more clumps. Place all the tubes containing samples 1-6 into a heat block at 95C for 5 min. You will need: a mini-PROTEAN tank. Assembling the electrophoresis module A general diagram of the apparatus is shown to right. Using a benchtop microcentrifuge (refrigeration isn’t necessary). SDS. Remove tubes from the heat block and centrifuge the tubes for 2 min at 5. centrifuge sample 1 for 5 min at 5. After centrifuging you want to retain the pellet. Calculate the amount of 5x TGS buffer to use to make 500 ml of a solution that is 1x TGS. Prepare a counterbalance by pipetting 500 ul of water into a 1. Remove the liquid (spent media) and dispose of in the bleach waste container.7 ml microcentrifuge tube. Experimental procedure Preparing the SDS-PAGE gel samples Samples 1-6 were collected during the protein expression and protein purification labs. 2. gel #2 the 2 . To each of these tubes add the necessary amount of the 6x loading buffer (calculated in Prelab Calculation above) and pipette to mix. Sample 1 requires more preparation since it contains whole cells that were collected before harvesting the culture. cover with attached electrical leads.000 rpm. The loading buffer contains glycerol.Virtual Drug Screening stream Spring 2011 Equipment and materials Mini-PROTEAN electrophoresis tank and lid Power supply and leads TGS running buffer Bio-Rad precast polyacrylamide gel 100 l 6x gel (or sample) loading buffer Protein Samples #1-6 from Expression and Purification labs Molecular Weight standards Plastic container w/ lid Imperial protein stain Prelab calculations 1. You will need to obtain a 100 l aliquot of 6x gel loading buffer from the TA/mentor.000 rpm. DTT (a chemical that reduces disulfide bonds). refer to the document ‘SDS-PAGE using the miniPROTEAN’ in Equipment Instructions on Blackboard.

Rinse the tank. into the second well. Write down which sample is associated with which well. Using tape. and molecular weight standards. So. Cut along the dotted line at the bottom of the cassette with a razor blade and pull off the tape to expose the slot along bottom edge of the gel. load 7 l of the MW standards (get from mentor) – write down the name of the MW standard that you used. With the buffer level markings on the front of the tank. label your dish with Name and Date. Removing and staining the gel After the gel electrophoresis is done. The gels and buffer dam should rest firmly against the green gaskets. The gel is thin and somewhat fragile so handle with care or it will tear. 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 tank lid and pour off and discard the running buffer. Usually. Now. turn off the power supply and disconnect the leads. Obtain a precast gel from the TA/mentor and remove from the packaging. (you will need it for the post lab). Remove the comb by sliding straight up. aligning the color coded plugs and jacks and insert the electrical leads into the power supply. This will allow the samples to run better. You stop your gel when the blue line reaches a centimeter or two from the bottom of the gel. making sure that the red electrode jack matches the red marking on the inside of the tank. use the buffer dam for the second gel position. you can load 20 l of each protein sample into each of the remaining wells using a P20 micropipettor. 500 ml of 1x TGS buffer. lid and assembly with DI water and hang to dry. Place the lid on the tank. the first well is skipped to prevent gel ‘smiling’ – or distortion. Do not let it go too long or the low MW proteins may run off the bottom of the gel. Set the timer on the power supply to 25 minutes Set the voltage to 200 V and select run. If you are running a single gel. on Sample loading and power conditions Clear each lane by using a ~20 gauge needle and syringe. Take care not to puncture the gel with the tips.Virtual Drug Screening stream Spring 2011 clamping frame (electrode assembly). Verify that your gel is running by looking for rising gas bubbles. Insert the assembly into the tank. buffer dam. wash the gel three times for 5 minutes each time with ~100 ml of nanopure water (‘washing’ means letting it sit in the dish 3 . Slide the green arms of the clamping frame over the gels and lock into place. This is necessary to allow contact of the bottom of the gel with the outer buffer reservoir. 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. The gels come with a 10-well comb that is used to form the wells. Use a 10 µl pipettor and let it fall gently into the well and sink to the bottom. Check with the TA/mentor to confirm that you have assembled the frame correctly. Place the gel cassettes into the clamping frame with the shorter glass plates facing inward. 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 the front for 2 gels. the assembly will fit in the position at the back of the tank. You are now ready to load your samples into the wells. Remove the gel by floating it off the plate into a large plastic dish of nanopure water. prepared protein samples. To remove excess SDS which will inhibit the staining process.

Cover with Saran Wrap (or cellophane – more permanent). Questions that should be answered in the course of your Lab Report (estimating molecular weight. At one of the appointed times. Alternatively. Set temp to 75OC on Gradient cycle for 1. look up the Molecular Weight standard that you used. The other person will need to print out a picture of it. Turn on vacuum and start cycle.e. Post your image online to Wiki page. Label the Petri dish with your initials and the date. 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. You can check your gel by lifting the lid . Get as many wrinkles out as you can. Once dry. VDS. You should find an image similar to the one at the right.5 hrs. pour the used reagent into the container marked for used stain – this stain can used again. Then turn off the vacuum pump. Date. Make sure your bands are distinguishable in the image. sarah wrap. 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. Cut a piece of Whatman filter paper that is large enough to cover your gel plus extra room for labeling. Take a picture of your dried gel. and what the gel is – i. 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). Print this out for your lab notebook and post an 4 . Write Name. break the vacuum seal first by lifting the sealing gasket. Wet the paper and put your gel on top of this. purity and yield): On the internet. Place a 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. Add a sufficient volume of stain to completely cover the gel (~30 ml) and place on the orbital shaker for 1-1.Virtual Drug Screening stream Spring 2011 while on the orbital shaker). labeling pen (DO NOT write on the paper before drying – wait til after!) gel. Cover with another piece of saran wrap. Save a copy for yourself on webspace or a USB drive. You can trim around the paper that the gel is stuck to (but leave room for labeling). Then lay them on the drying bed. Dry the gel Materials: Whatman paper. Lay the sealing gasket over everything.A dry gel should be warm over the entire surface. Flip a coin to determine who gets to paste the actual dried gel in their Lab Notebook. On the front of the paper. When finished with the staining. take your gel over to the Biotech lab to be dried so that you can save it and put in your notebook. use the flat bed scanner. Lay a piece of saran wrap on the flat bed scanner then flip your gel on top – so it is face down.5 hours until dark bands are very visible. label what is in each lane.

Results: show images of your results that you have already taken Transformation plates images . address sources of error.html End with a transition statement where you briefly sum-up what was done in these labs and its importance. No need for a hypothesis in this write up. Now. purification.de/pepcore/pepcore_services/index. There is a good article here: Nat Methods.Virtual Drug Screening stream Spring 2011 electronic copy for your Wiki Lab Report.250 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 information on protein production and purification. estimate the MW of your purified protein using the standards as a reference.embl. Protein production and purification. 2008 Feb. M&M 250 words: be succinct while allowing an informed reader to be able to replicate your work. Nanodrop screen shots . the estimated purity would be 50%. Also a good website: http://www. 1. Do not show calculations for prelabs. Show calculations for Beer’s law calculatins.Mention how many colonies you got.5(2):135-46. and characterization. answer any questions from the handout Conclusions References 5 . Flask images. Image of Ladder (molecular weight marker used) Discusssion Analyze your results. Examples of Bio-Rad Molecular Weight Standards How many other protein bands are present in the sample 5 lane? If there was one other band of equal intensity. Protein Gel images. B checks in the morning!) This report will encompass the last three (3) labs: protein expression.Include yields from your Nanodrop spectrophotometry for your protein amounts. 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: Due Monday April 18th at Midnight (or before Dr. purple pellet images (include the weight of your pellet) Images of Elution 1 and 2 in tubes.