1、凝胶层析凝胶层析 Gel Filtration Chromatography Bio96 zoumi 2009030069 1.Objectives 1)To understand the principle of gel chromatography and its application.2)By measuring the protein molecular weight training,initially master gel chromatography.2.principles Gel chromatography,also known as exclusion chromato
2、graphy,gel filtration,molecular sieve chromatography,filtration or infiltration.It is widely used in separation,Purification,concentration and desalting of biological macromolecules,and so on,while the determination of protein molecular weight is also one of its important applications.Gel is a kind
3、of three-dimensional network structure was porous and the bead-shaped insoluble particulate material.When using it to separate substances,it is mainly according to the different radii of protein molecules(near-spherical)with different exclusion effects.That is,it is based on the molecular size of th
4、e physical properties of the separation and purification.For certain types of gel,some molecules cannot enter the particles,and can only flow along the outer column;while a number of small molecules are not excluded.They are free to spread,infiltrating into the gel inside the sieve,and later is take
5、n out.The smaller molecules,the deeper inside into the gel,the more distance they go,so the small molecules are the last to flow out.Some medium-sized molecules between macromolecules and small molecules can only enter the part of the larger gel pores,which is partly excluded,so these molecules come
6、 out between large and small molecules.This sample after gel chromatography,the molecules will be in accordance with both large and small in the flowing order to achieve the purpose of separation.For any kind of compounds separated by gel,the exclusion chromatography columns are in the range of 0 10
7、0%,and its ranked degree of being excluded can be described by Kav(isolated compounds,including outer water and external water volume in the proportional relationship).As for K av and the total volume of gel column bed(V t),outer water volume(V 0),and the elution volume(V e)there is a formula like t
8、his:K a v=(V e-V 0)/(V t-V 0).Chromatography under the fixed conditions,V t and V 0 is a constant value,V e changes with the changes of the mass of the molecules to be separated.The larger the molecular weight is,the smaller the smaller V e value and Kav value will be.On the contrary,the smaller the
9、 molecular is,the bigger the V e value and K av value will be.Kav is important to estimate the separation effect and the mass of protein molecules.In the same chromatographic conditions,the more different the K av values of separated material is,the better separation effect will be.On the contrary,s
10、eparation efficiency,otherwise the molecules cant be separated or the effect is very bad.In the actual experiment,we measured a V t,V 0 and V e values,and then calculate the K av.For a particular type of gel,in the context of a certain molecular weight,K av,and lg M r(M r indicated that substance mo
11、lecular weight)into a linear relationship:K av=-blg M r+C The same can be:V e=-b lg M r+C Where b,C are constant.Namely,V e and lg M r has a linear relationship.We can isolate Known-weight molecular proteins in a variety of gel column,and in accordance with the above-mentioned linear relationship be
12、tween the plotted standard curves,and then measure the other unknown protein molecular weight using the same gel column.3.Equipment and reagents 1)Equipment (1)Glass chromatography (2)DHL-B PC constant pump(Shanghai Jing Branched Industrial Co.,Ltd.)(3)BSZ-100 automatic part of the collector(Shangha
13、i Qingpu Instrument Factory Huxi)(4)8823B UV detector (5)UNICCO UV-2102 C uv-vis spectrophotometer (6)TYPE3057 Portable Recorder(Sichuan No.4 Instrument Factory)(7)TB-600 gradient mixer(Shanghai Unity Biochemical Instrument Factory)(8)100ml reagent bottle (9)1000ml graduated cylinder (10)250ml beake
14、r (11)50ml,100ml beaker (12)10ml test-tube scale 2)Reagents Standard protein Bovine serum albumin:M r=67 000(Shanghai Biochemistry).Egg albumen protein:M r=45 000(U.S.SIGMA company).Lysozyme:M r=14 300.Unknown protein samples Elute 0.025mol/L KCL 0.1 mol/L HAC Blue dextran 2000 4.Experiment procedur
15、es 1)Gel swelling Weigh 7g Sephadex G-75 in the 250 ml beaker,add 100ml elate,swell at room temperature for 2 3d,repeatedly Pour remove the fine particles,and then place gel in vacuum to cast the air between the particles,then into boiling water bath to boil 2 3h(to run away Internal air of the part
16、icles,and to sterilized).2)Loading the column (1)Take a clean glass chromatography column and vertically fixe it to the metal framework stage.(2)Determining the total volume of gel column(V t)Make a mark 5cm away up to the column end,close the column outlet,add deionized water,open the outlet.When t
17、he liquid level drop to the mark,close the outlet.Then use the measuring cylinder to accept the de-ionized water(surface down to the glass sieve),read the column.Out of the total volume of the column bed volume shall be V t.We can also measuring V t after having done the unknown-protein.(3)adding el
18、ate to the column(about 1/3 column bed height),slowly pour t thick slurry to the column until the gel becomes 1cm 2cm high and then open the outlet,the flow rate tends to be about 3 6ml/10min.when the gel goes to the mark,and the loading is completed,be careful that the loading gel cannot be layered
19、.And then close the outlet,put it aside for a moment,until the gel deposits,then connect it to the constant-current.Use 1 2 times of the bed volume of elate to balance column,so that make sure of the stability.3)Determining V 0 and the V e of the unknown protein Draw away the elate on the surface(mu
20、st not upset the surface,filter paper or nylon net can be covered).Open the outlet,so that residual liquid is reduced to glue facial cut(but not let it dry).Close the outlet.Suck up with a fine dropper 0.5ml mixture of(4.5mg/ml)blue-color-glucan-2000 and unknown protein(5mg/ml)mixture,carefully add
21、it circling around the column wall(2mm above the gel surface),and then open the outlet(Start collecting!),After the solution into the gel bed,Close the outlet with a little eluent adding to the bed,fill the top with eluent.Start eluting at the speed of 5 6ml/10min.automatic collector for each tube i
22、s set at 5min collection.Finally make elution curve Finally made elution curve.Collection and planning based on expenditure from the Canadian Kind of beginning to the elution concentration of blue dextran elution volume shall be the highest point of the V 0.Note:Blue Dextran wash down Afterwards,use
23、 the elate(1 2 times the bed volume)to balance,preparing for the next step 4)The standard curve (1)The standard protein solution with the eluent is prepared for class use,the solution of various concentrations of the three proteins are:bovine serum Albumin(5mg/ml),chicken egg albumin(8mg/ml),lysozym
24、e(4.5mg/ml).(2)According to the operation method(3)add the standard protein 0.5 ml of the solution 5 6ml/10min and collect eluent.(3)By using ultraviolet spectrophotometer determine A280 tube by tube and decide the peak point of protein elution,and then measured protein elution volume Ve.Note that a
25、fter the experiment is completed,get all the gel I recycled for the next test to use.Its strictly prohibited to discard,or pour the gel into the Pool.5.Records 1)raw data Elute Speed:0.6ml/min Automated portion of the collector speed:(for the first time)5 min/tube,(second)3min/tube Recording Papers
26、speed:2 cm/h UV Detector:(for the first time)0.5A,(second)0.2A The total volume of gel columns:V t=86.0ml The blue dextran elution volume(external water volume):V 0=31.3ml Unknown protein elution volume:V g=51.4ml Standard proteins:V e1=32.2ml V e2=41.2ml V e3=65.6ml 2)Absorbance A280 Data number A2
27、80 V Number A280 V 1 0 16 0.093 2 0.002 17 0.080 3 0.002 18 0.124 4 0.008 19 0.274 5 0.003 20 0.454 6 0.003 21 0.531 51.4ml 7-0.003 22 0.436 8-0.003 23 0.266 9 0.001 24 0.125 10 0.011 25 0.026 11 0.001 26 0.000 12 0.397 27-0.017 13 1.170 31.3ml 14 0.590 15 0.188 Table 2-1 280nm UV absorbance of samp
28、le 1 number A280 V number A280 V 1 0.000 26 0.059 2 0.001 27 0.065 3-0.001 28-0.022 4-0.003 29-0.048 5-0.002 30-0.049 6 0.003 31 0.047 7 0.001 32 0.025 8 0.002 33 0.102 9-0.001 34 0.184 10-0.001 35 0.251 11-0.001 36 0.272 12 0.001 37 0.276 65.6ml 13 0.002 38 0.2 14 0.011 39 0.131 15 0.004 40 0.059 1
29、6 0.011 41 0.101 17 0.142 42 0.07 18 0.206 32.2ml 43 0.123 19 0.173 44 0.031 20 0.141 45 0.019 21 0.124 46 0.004 22 0.082 47 0.083 23 0.165 41.2ml 48 0.019 24 0.129 49 0.005 25 0.116 50 0.003 Table 2-2 280nm UV absorbance of sample2 标准蛋白洗脱曲线 Figure2-1 Standard protein elution curve 6.Data Processing
30、 1)The total volume of gel column V t=86.0ml 2)External water volume V 0 and the unknown protein elution volume V e Figure 2-2Absorbance Graph of sample 1 The first elution peak is for the external water volume V 0.The second peak is for unknown protein V e.Between the column and the automatical col
31、lector,there is about 5ml liquid.When count the elution volume,it should be subtracted.V 0=36.0-5.0=31.0(ml);V unknown=51.4-5.0=46.4ml.3)Standard protein elution volume V e Figure 2-3 Absorbance Graph of sample2 The first three peaks are as following:bovine serum albumin elution peak,egg albumin elu
32、tion peak,lysozyme elution peak.Bovine serum albumin:M r=67 000,albumin,:M r=45 000,Lysozyme:M r=14 300.Measured:V e(bovine serum albumin)=32.2-5.0=27.2ml;V e(egg albumin)=41.2-5.0=36.5ml;V e(lysozyme)=65.6-5.0=60.6ml 4)The protein in the effective distribution coefficient K av and the unknown prote
33、in molecular weight calculation protein Mr LgMr Vt/ml V0/ml Ve/ml Kav bovine serum albumin 67000 4.826 86.0 26.3 27.2 0.015 egg albumin 45000 4.653 36.5 0.171 lysozyme 14300 4.155 60.6 0.575 unknown protein 46.4 0.337 Figure2-4 Kav-lgMr gragh of the known proteins Put the unknown protein K av=0.337 into the fitting formula.So we can fet lgMr=4.311,Mr=27803.53.Figure2-5 Ve-lgMr gragh Get the unknow
