BCOR11 Laboratory6: Proteins Part II Separation of Protein Extracts using PA E Out!ine of Acti"ities an# Ob$ecti"es I% Separation of Protein Sa&p!

!es by PA E 'Intro#uction to #iscontinuous po!yacry!a&i#e ge!s ' ain practice (or)ing (it* po!yacry!a&i#e ge!s 'Prepare sa&p!es base# on protein concentrations #eter&ine# in Lab + 'Practice !oa#ing, running, an# staining po!yacry!a&i#e ge!s 'As ti&e per&its, stu#ents (i!! practice &a)ing a ge! using ge!atin II% -n#erstan#ing Intro#uctions '.e&onstrate )no(!e#ge of t*e purpose of intro#uctions an# i&portant points to consi#er (*en (riting '.iscuss expectations for t*e Intro#uction section you (i!! (rite

In Lab 5, you discussed the concept of PAGE, prepared protein samples, and determined their concentrations using a Bradford Assay. Today, you ill be able to separate your protein samples by PAGE to gain practice in the techni!ue and to compare and contrast the proteins found in samples isolated from different sources "fish, egg hite, and li#er$.

Separation of Protein Sa&p!es by Po!yacry!a&i#e e! E!ectrop*oresis /PA E0 To separate your samples, you ill use a polyacrylamide gel consisting of a top gel hich is lo in acrylamide concentration and a bottom gel hich is higher in concentration of acrylamide. These polyacrylamide gels ha#e t o parts that contain different percentages of polyacrylamide in order to obtain crisp separation of the protein bands. The bottom portion, hich ma%es up the ma&ority of the gel, is called the resol#ing gel and the top part is called the stac%ing gel. Typically, stac%ing gels contain a lo percentage of polyacrylamide "'()$, and thus ha#e a fairly loose matri*. +esol#ing gels ha#e a higher percentage of polyacrylamide, and therefore tighter matri*, that #aries depending on the si,e of proteins you ish to separate and ho far you ant to separate them. -ost separating gels are bet een .) and /5) polyacrylamide. 0o you thin% that proteins mo#e more !uic%ly through the stac%ing or the separating portion of a gel1 2hat do you thin% the purpose of the stac%ing gel is1 If you anted to separate a sample containing #ery large proteins, do you thin% it ould be best to use an .) or a /5) separating gel1 2hy1 Polyacrylamide Gel Preparation 33AL2A45 2EA+ GL67E5 28ILE 26+9I:G 2IT8 P6L4A;+4LA-I0E33 /$ +esol#ing gel "prepared for you by the laboratory coordinator$ A /<) resol#ing gel has already been poured for you using the recipe and procedure belo . =or t o resol#ing gels "'/> mls$ Reagent Volume distilled ater ?.?5 mls
1

resol#ing buffer, p8 ..@ acrylamideBbis Temed />) ammonium persulfate

<.A mls (.> mls 5.> Cl 5>.> Cl

- Add all components e*cept Temed and ammonium persulfate to a 5> ml bea%er. - Add Temed and ammonium persulfate and pour gels immediately. Temed and ammonium persulfate polymeri,e the polyacrylamide in solution to form a solid matri*, so it is important to pour the gels !uic%ly after adding them. - ;arefully add a layer of ater on top of the gel to pre#ent it from drying out. If desired, the gel can be rapped ith parafilm and stored until the follo ing day. <$ 5tac%ing gel - +emo#e any ater from the top of the gel. - Prepare the () stac%ing gel as follo s. =or t o stac%ing gels "'(.> mls$ Reagent d8<6 Tris buffer, p8 D.. acrylamideBbis Temed />) ammonium persulfate Volume <.5 mls /.> mls >.5 mls 5.> El 5>.> El

- ;ombine all reagents e*cept Temed and ammonium persulfate in a <5 ml bea%er. -i* gently by s irling. - Add Temed and ammonium persulfate !uic%ly. ;arefully s irl to mi*. - -a%e sure ater is remo#ed from top of the resol#ing gel - Immediately layer the stac%ing gel onto the resol#ing gel using a pipette and applying the acrylamide mi*ture to one corner of the gel. - Insert the comb gently to a#oid air bubbles. Any air bubbles ill pre#ent polymeri,ation of the gel. - Allo ?> minutes for the gel to polymeri,e. Protein 5ample Preparation "complete hile aiting for your gel to polymeri,e$ /$ Fsing the protein concentrations obtained from your Bradford assay "Lab 5$ from the samples you ha#e fro,en a ay, perform the calculations necessary to determine hat #olume of your sample you need to use to load bet een <> and 5> Eg of protein on your gel. =ill in the table belo . The total #olume of each sample should be <5 El. =or <5 El samples Sample Concentration Volume (for 20-50 g) Laemmli Sample Buffer* ( El
2

dH2 (to 25 L total)

( El ( El 3 Laemmli sample buffer contains 505, glycerol for helping the sample to Gsin%H into the ell of the gel, and a blue dye for trac%ing the protein sample as it tra#els in the gel. <$ 6btain three microfuge tubes and add the appropriate #olumes of sample, ater, and Laemmli sample buffer. Be sure to label your tubes. ?$ Get a microfuge tube of molecular eight mar%ers from the free,er. -olecular eight mar%ers contain proteins of specific, %no n molecular eights in order to help identify the molecular eights of proteins in your samples. The mar%ers are GprestainedH "so you can see them as your gel is running$ and already contain sample buffer. 4ou ill only need to load /> El onto your gel. !our "# $ill gi%e &ou a picture $it' t'e information on protein( contained in t'e molecular $eig't (tandard() Protein 5eparation /$ 6nce your gels ha#e polymeri,ed completely, gently remo#e the combs by pulling straight up. 4ou ill notice that you ha#e /5 lanes on each gel. 5et up your gel as sho n in the diagram belo . There ill probably be t o gels for the entire class. <$ Load your protein samples and the molecular eight mar%ers in indi#idual lanes. Fse one lane for the molecular eight mar%er for each gel. T o gels ill probably be re!uired for the entire lab section. ?$ In your noteboo%, diagram the gel and indicate the contents of each lane. This is importantI 2hy1 ($ +un your gel at />> 7olts for (5 minutes. 4ou can follo ho the proteins ha#e migrated through the gel by obser#ing the blue dye in the Laemmli sample buffer. 2hile your gel runs, you ill discuss Introduction sections "see belo $. 5$ 5tain your gels - 2hen the gel has finished running, your laboratory TA ill remo#e it from the apparatus. - To be able to #isuali,e the proteins in your samples, stain it for at least ?> minutes ith ;oomassie Blue "a dye that binds to proteins$. If necessary, the gel can be left o#ernight in ;oomassie Blue. 2hen staining is finished, remo#e and sa#e the ;oomassie dye in the container in the hood to be reused. - To get rid of e*cess dye, the gel ill be placed in destain "(>) methanol, A.5) acetic acid$ until the bac%ground is clear and the protein bands are #isible. - 4our laboratory TA ill eJmail you a picture of your gel, and you ill discuss the results at the beginning of Lab A.
3

0iagram for 5etting Fp Polyacrylamide Gels

6n the right is an e*ample of hat your gel might loo% li%e, along ith the data for the molecular eight mar%ersK

Appen#ix (>) Acrylamide 5toc% ?..@) Acrylamide /./) bisacrylamide bring to />> ml total #olume ith d8<6 sterile filter and store at (L; in a lightJproof container (M Polyacrylamide +esol#ing Gel Buffer /.5 - Tris base >.() 505 p8 to ..@ and bring to />> ml total #olume ith d8<6 sterile filter and store at (L; (M Polyacrylamide 5tac%ing Gel Buffer >.5 - Tris base >.() 505 p8 to D.. and bring to />> ml total #olume ith d8<6 sterile filter and store at (L; DM Laemmli Protein 5ample Buffer >.?A5 - TrisJ8;l, p8 D.. /<) 505 ?>) NJmercaptoethanol D>) glycerol >.>/<) Bromophenol Blue +unning Buffer for Polyacrylamide Gel Electrophoresis <.A? g Tris base /<..( g glycine >.@ g 505 bring to (5> ml total #olume ith d8<6 ;oomassie Blue 5tain <>> ml methanol 5> ml acetic acid 5> ml /.> g ;oomassie blue bring to 5>> ml total #olume ith d8<6 ;oomassie Blue 0estain <>> ml methanol A5 ml acetic acid bring to />>> ml total #olume ith d8<6
5

-n#erstan#ing Intro#uctions 4ou ha#e already discussed and completed t o sections of a laboratory reportK -aterials O -ethods and +esults. =or the upcoming bacterial transformation labs, you ill rite Introduction and 0iscussion sections as ell as -aterials O -ethods and +esults sections. 4ou ill tal% about Introductions in todayPs lab and 0iscussions during Lab @. In preparation for todayPs discussion you read about riting Introductions and completed a preJlab assignment. As a class, you ill discuss the ans ers to the preJlab assignment !uestions, general tips for riting Introductions, and any specific e*pectations your laboratory TA has for your Introductions. The main goals of an Introduction are to /$ capture the attention of the reader, <$ to gi#e them the bac%ground %no ledge they need to be able to understand the purpose, results, and implications of your or%, and ?$ e*plain hy you are riting your report. -ost Introductions begin by gi#ing the reader the Gbig picture,H describing in fairly general terms hat is %no n about the topic under in#estigation. An Introduction should not describe the specific details of the e*periments that led to this %no ledge, but rather present the main findings and conclusions of other researchers "properly referenced, of courseI$. The best Introductions stri%e a balance bet een gi#ing the reader too many details or pro#iding too little information. +emember that you ant to ma%e sure the reader is able to fully understand your study, but not to o#er helm them ith irrele#ant details. It is also important to %eep in mind your intended audience "i.e. other researchers in your specific field, or a more general audience$ hen thin%ing about the appropriate le#el of detail to include. In addition to the necessary bac%ground information, an Introduction should tell the reader $'& you performed your analyses. 4ou should e*plain ho the !uestion you addressed relates to hat is %no n in the field and hat ne information your analyses attempted to obtain. 2hat as the main goal of your study1 2hy is your !uestion important to ans er "i.e. ho can it add to hat is %no n about your topic$1 -ost introductions conclude by ith a #ery brief description of the %ey findings and conclusions to Gset the stageH for the +esults and 0iscussion sections. 6#erall, the Introduction is your chance to grab your readerPs attention and ma%e them ant to continue reading your paper.

BCOR11 Laboratory Exercise 6 Post'Lab Assign&ent .ue at t*e beginning of Lab 1 Ans er the follo ing !uestions completely, but succinctly "'/J? sentences each$. Please type or neatly rite your ans ers on a separate sheet and number the !uestions appropriately. Be (ure to put &our name* la+ (ection* and t'e name of &our la+orator& "# on &our a((ignment) This assignment must be completed by t'e +eginning of t'e ne,t la+ period (La+ -).

/$ 2hat is the purpose of a stac%ing gel1

<$ If you anted to separate proteins that ere #ery small, ould it be better to use a higher or lo er percentage resol#ing gel1 2hy1

?$ 2hy does the Laemmli protein sample buffer contain 5051 "8int Q if you canPt remember, loo% bac% at Lab 5$

($ 2hen you ran your gel, the proteins migrated from the ells at the top of the gel to ards the bottom of the gel. 2hy1 "8int Q it may help to refer to the diagram sho ing ho set up a polyacrylamide gel$

5$ 2hat is the si,e range of the molecular eight mar%ers you used "i.e. hat is the smallest protein and hat is the biggest protein$1 2hich mar%er as closest to the bottom hen you stopped running your gel1

D$ 2hat do you notice about the ay the mar%ers are separated on the gel1

In preparation for your discussion of the results, you should loo% at the picture of your gel that your laboratory TA ill email to you and t'in. about the follo ing !uestions "you do not ha#e to turn in your ans ers, &ust be prepared to discuss the !uestions$. - 8o did your gel run1 2hat si,es of proteins are found at the top of the gel1 At the bottom1 - =rom your %no ledge of the molecular eight mar%ers, can you tentati#ely identify any of the protein bands in the samples1 - 0o the protein samples appear to be related hen you obser#e their electrophoretic pattern1 - 2hat are the limitations of ;oomassie staining as a techni!ue to #isuali,e proteins1