各种缓冲液.pdf

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1、STOCK SOLUTION RECIPIES:STOCK SOLUTION RECIPIES:Tris-HCl BufferTris-HCl Buffer10X Tris-HCl (0.5M Tris Base, pH7.6): Trizma Base - 61 g Distilled water - 1000 ml Adjust pH 7.6 using concentrated HCl Store this solution at room temperature. Dilute 1:10 with distilled water before useand adjust pH if n

2、ecessary.20X Tris-HCl (1M Tris Base, pH7.6): Trizma Base - 122 g Distilled water - 1000 ml Adjust pH 7.6 using concentrated HCl Store this solution at room temperature. Dilute 1:20 with distilled water before useand adjust pH if necessary.10X Tris-HCl-Tween 20 (0.5M Tris Base, 0.5% Tween 20, pH7.6):

3、 Trizma Base - 61 g Distilled water - 1000 ml Adjust pH 7.6 using concentrated HCl and then add 5 ml of Tween 20. Store this solution at room temperature. Dilute 1:10 with distilled water before useand adjust pH if necessary.Note: Tris-HCl Buffer is used for specific cases of immunohistochemical sta

4、ining.* OR you can use Tris Base to make Tris-HCl (note that Tris base is different OR you can use Tris Base to make Tris-HCl (note that Tris base is differentfrom Trizma)from Trizma)Tris is a chemical with basic properties, having a pKa of 8.1. It can be used to buffersolutions from drastic pH chan

5、ges, keeping them in the pH range of 7.0 to 9.0.Make any Tris-HCl buffer in this pH range, at any molarity using these simple steps1) Calculate Moles of Tris Basemol/L * L = moles needed2) Calculate Mass of Tris BaseDetermine the mass of Tris base to weigh by multiplying the number of moles bythe mo

6、lecular weight (121.14 g/mol) of Tris.moles needed * g/mol = g3) Dissolve Tris Base in WaterDissolve the required mass of Tris into a volume of deionized waterapproximately 1/3 of the desired volume of buffer to be made.4) Adjust the pHUsing a pH meter, titrate the solution of Tris with 1M hydrochlo

7、ric acid (HCl)until the correct pH is reached.5) Bring to VolumeAdd the TrisHCl mixture to a volumetric flask of the desired volume and adddeionized water as required to complete the solution.0.2M Phosphate Buffer-4 Liters (pH 7.4):0.2M Phosphate Buffer-4 Liters (pH 7.4):17.66g Sodium Phosphate Mono

8、basic90.03g Sodium Phosphate Dibasic Heptahydrate4 Liters ddH2OpH should be 7.4, if not adjust with 1.0N NaOH or 1.0N HCl (3 Liters-13.25g Mono and 67.52g Dibasic)0.1M Phosphate Buffer Saline (PBS)-8 Liters:0.1M Phosphate Buffer Saline (PBS)-8 Liters:Prepare 4 liters of 0.2M phosphate buffer (see ab

9、ove recipe)Add 72g NaCl (0.9% or 9g/liter)Add 4 liters of ddH2OpH=7.4*For practical purposes, you can also make 16 liters of PBS by first preparing 4 liters of0.4M Phosphate Buffer. This concentration uses twice as much Monobasic and Dibasicsince 0.4M versus 0.2M means the solution is twice as conce

10、ntrated. But remember, youmust then dilute this solution by adding 12 liters of water to make a total of 16 liters at aconcentration of 0.1M. Also, since we are using 9gNaCl/liter, a total of 144g of NaClwill be used.0.1M Phosphate Buffer-1 liter:0.1M Phosphate Buffer-1 liter:0.5 Liter of 0.2 M Phop

11、hate Buffer Stock0.5 Liter of ddH2O0.1M Phosphate Buffered Saline with Azide-1 liter (PBS*):0.1M Phosphate Buffered Saline with Azide-1 liter (PBS*):Prepare one liter of 0.1M Phosphate Buffer (see recipe above)Add 9g of NaClAdd 200mg of sodium azide (contents of 1 eppendorf vial)Add 0.3 ml Triton-X*

12、Be sure to use protective clothing and a mask when handling sodium azide under hood!1% Paraformaldehyde 2% Glutaraldehyde1% Paraformaldehyde 2% Glutaraldehyde0.2M Stock Phosphate Buffer pH 7.4 500mlParaformaldehyde 10gGlutaraldehyde (25% in water) 80mlDistilledwater QS to 1000mlDissolve the paraform

13、aldehyde in about 400 ml of water. Heat this solution to 58 to 60degrees C(do not allow the solution to get to hot!). Add 1N NaOH drop by dropuntil thesolution turns clear. Add the phosphate buffer stock and allow to cool, and then add theglutaraldehyde. Ph the solution to 7.4 and FILTER before use.

14、1% Paraformaldehyde 2% Glutaraldehyde (Hrp Fix)1% Paraformaldehyde 2% Glutaraldehyde (Hrp Fix)0.2M Stock Phosphate Buffer pH 7.4 500mlParaformaldehyde 10gGlutaraldehyde (25% in water) 80mlDistilled water QS to 1000mlDissolve the paraformaldehyde in about 400ml of water. Heat the solutionto 58 to 60d

15、egrees C (do not allow the solution to get too hot!). Add 1N NaOH drop by dropuntilthe solution clears.Add phosphate buffer stock and allow the solution to cool. Addtheglutaraldehyde and FILTER.2% PARAFORMALDEHYDE2% PARAFORMALDEHYDE0.2M Stock Phosphate Buffer pH 7.4 500mlParaformaldehyde 20gDistille

16、d water QS to 1000mlDissolve the paraformaldehyde in about 400ml of water. Heat the solutionto 58 to 60degrees C(do not allow the solution to get too hot!). Add 1N NaOH drop by dropuntilthe solution clears.Add phophate buffer stock and allow the solution to cool. pH to 7.4and FILTER.4% PARAFORMALDEH

17、YDE4% PARAFORMALDEHYDE0.2 M Stock Phosphate Buffer pH 7.4 500mlParaformaldehyde 40gDistilled water QS to 1000mlDissolve the paraformaldehyde in about 400ml of water. Heat the solution to 58 to 60degrees C(do not allow the solution to get too hot!) Add 1N NaOH to theparaformaldehyde solution drop by

18、drop until the solution clears. Add phosphate bufferstock and allow to cool. pH to 7.4 and FILTER.4% PARAFORMALDEHYDE 0.2% GLUTARALDEHYDE4% PARAFORMALDEHYDE 0.2% GLUTARALDEHYDE0.2M Stock Phosphate Buffer pH 7.4 500mlParaformaldehyde 40gGlutaraldehyde (25% in water) 8mlDistilled water QS to 1000mlDis

19、solve the paraformaldehyde in about 400 ml of water. Heat the solutionto 58 to 60C(do not allow the solution to get too hot!). Add 1N NaOH drop by dropuntil thesolution clears. Add phosphate buffer stock and allow the solution to cool. Add theglutaraldehyde. pH to 7.4 and FILTER.Sucrose solutionSucr

20、ose solution10% 10gram in 90 ml 0.1 M PB20% 20gram in 80 ml 0.1 M PB30% 30gram in 70 ml 0.1 M PBAcrylamide for separating gelAcrylamide for separating gel (Acrylamide : BIS = 30 : 0.135) Acrylamide 30.00 g BIS 0.135 g Make volume to 100 ml with MQ water. Keep in dark (Brown bottle)Separating gel buf

21、fer (pH 8.8)Separating gel buffer (pH 8.8) (Final Conc.) Tris 12.11 g 1 M SDS 0.27 g 0.27% Dissolve in 80 ml MQ water, adjust pH to 8.8, make the vol. to 100 mlAcrylamide for stacking gelAcrylamide for stacking gel (Acrylamide : BIS = 29.2 : 0.8) Acrylamide 29.2 g BIS 0.8 g Make volume to 100 ml wit

22、h MQ water. Keep in dark (Brown bottle)Stacking gel buffer (pH 6.8)Stacking gel buffer (pH 6.8) (Final Conc.) Tris 3.03 0.25 M SDS 0.20 g 0.2% Dissolve in 80 ml MQ water, adjust pH to 6.8, make the vol. to 100 mlSDS-PAGE running bufferSDS-PAGE running buffer Tris 9.0 g Glycine 43.2 g SDS 3.0 g Disso

23、lve in 3 L MQ waterSDS-sample bufferSDS-sample buffer Glycerol 10 ml Tris 0.757 g SDS 2.5 g 2-Mercaptoethanol 5.0 ml Dissolve in 100 ml MQ waterBromophenol blue (BPB) solutionBromophenol blue (BPB) solution Dissolve 0.1 g BPB in 100 ml 10% glycerolBlotting solution ABlotting solution A (Final conc.)

24、 Tris 36.33 g 0.3 M Methanol 200 ml 20% SDS 0.20 g 0.02% Make vol to 1000 ml with MQ water, keep at 4 CBlotting solution BBlotting solution B (Final conc.) Tris 3.03 g 25 mM Methanol 200 ml 20% SDS 0.20 g 0.02% Make vol to 1000 ml with MQ water, keep at 4 CBlotting solution CBlotting solution C (Fin

25、al conc.) Tris 3.03 g 25 mM s-Amino-n-Caproic Acid 5.20 g 40 mM Methanol 200 ml 20% SDS 0.20 g 0.02% Make vol to 1000 ml with MQ water, keep at 4 CPonceau 3S staining solutionPonceau 3S staining solutionDissolve 0.1 g Ponceau 3S in 100 ml 1% Acetic acidCoomassie brilliant blue (CBB) solutionCoomassi

26、e brilliant blue (CBB) solution Methanol 1500 ml Acetic acid 300 ml Coomassie brilliant blue-R250 3 g Dissolve in 3000 ml MQ waterDestaining solutionDestaining solution Methanol 1100 ml Acetic acid 300 ml Dissolve in 3000 ml MQ waterAcrylamide solution for 1D-PAGEAcrylamide solution for 1D-PAGE(Acry

27、lamide : BIS = 28.38 : 1.62) Acrylamide(High grade) 28.38 g BIS 1.62 g Make vol to 1000 ml with MQ water, keep in darkLysis buffer for Western BlotLysis buffer for Western Blot Lysis buffer stock 1 ml Tris HCl, pH 7.4 (pre-made stock) 0.584g NaCl 49 ml dH2O This stock can be kept for up to 2 weeks a

28、t 4 C Lysis buffer for tissue prep 10 ml lysis buffer stock (see above) 1 protease inhibitor tablet 0.2 ml Phosphatase inhibitors (1 mM Na3VO4 and 5 mM NaF to block both tyrosineand serine/threonine kinases, see below) 100 ml (50 x Na3VO4 and NaF stock solution) 50 mM Na3VO4 0.92gl 500 mM NaF 2.0995

29、g 100 ml dH2O0.02 N H0.02 N H3 3POPO4 4 Add 0.48 ml H3PO4 (42 N, specific weight 1.87, 72%) to 1000 ml MQ water0.02 N NaOH0.02 N NaOH Dissolve 0.8 g NaOH to 1000 ml MQ buffer (RadioImmunoPrecipitation Assay) buffer:RIPA buffer (RadioImmunoPrecipitation Assay) buffer:RIPA buffer contains the ionic de

30、tergent sodium deoxycholate as an active constituentand is particularly used for nuclear membrane disruption for nuclear extracts. A RIPAbuffer gives low background but can denature kinases. It can also disrupt protein-proteininteractions (and may therefore be problematic forimmunoprecipitations/pul

31、l down assays).50mM Tris HCl pH 8150 mM NaCl1% NP-400.5% sodium Deoxycholate0.1% SDSThe 10% sodium deoxycholate stock solution (5 g into 50 ml) must be protected fromlight.The 100 mM EDTA stock solution is made with 1.86 g into 40 ml H2O and then addNaOH to dissolve and adjust pH to 7.4. Finally, ad

32、just the total volume to 50 ml). Storethe buffer at 4C.Nonidet-P40 (NP-40) buffer:Nonidet-P40 (NP-40) buffer:20 mM Tris HCl pH 8137 mM NaCl10% glycerol1% nonidet P-402 mM EDTASodium orthovanadate preparation:Sodium orthovanadate preparation:This needs to be done under the fume hood Prepare a 100 mM

33、solution in double distilled water Set pH to 9.0 with HCl Boil until colorless Cool to room temperature Set pH to 9.0 again Boil again until colorless Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling Bring up to the initial volume with water Store in aliquots at -20CN

34、ote: do not permit great changes in volume during boiling; put a loose lid on thecontainer to protect from evaporation. Discard if the samples turn yellow.TBS 10 x (concentrated TBS)TBS 10 x (concentrated TBS)24.23 g Trizma HCl80.06 g NaClMix in 800 ml ultra pure water.pH to 7.6 with pure HCl.Top up

35、 to 1 L.TBSTTBSTFor 1 L:100 ml of TBS 10 x+ 900 ml ultra pure water+ 1ml Tween20Medium stripping buffer:Medium stripping buffer:Make fresh stripping buffer:15 g glycine1 g SDS10 ml Tween20Set the pH to 2.2make up to 1 L with ultrapure waterHarsh stripping buffer:Harsh stripping buffer:to be done und

36、er the fumehoodFor 100 ml:20 ml SDS 10%12.5 ml Tris HCl pH 6.8 0.5M67.5 ml ultra pure waterAdd 0.8ml -mercaptoethanol under the fumehood.Nuclear Fractionation Protocol ReagentsNuclear Fractionation Protocol ReagentsBuffer ABuffer A 10 mM HEPES,1.5 mM MgCl2,10 mM KCl,0.5 mM DTT,0.05% NP40 (or 0.05% I

37、gepal or Tergitol)pH 7.9To prepare 250 ml stock of buffer A250 ml stock of buffer A HEPES: 1M = 238.3 g/L, therefore 10 mM = 0.59 g/250 mlMgCl2: 1M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mlKCl: 1M = 74.5 g/L, therefore 10 mM = 0.187 g/250 mlDTT: 1M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 ml

38、NP40 = 0.05%Buffer BBuffer B 5 mM HEPES,1.5 mM MgCl2,0.2 mM EDTA,0.5 mM DTT,26% glycerol (v/v)pH 7.9To prepare 250 ml stock of buffer B 250 ml stock of buffer B HEPES: 1M = 238.3 g/L, therefore 5 mM = 0.295 g/250 mlMgCl2: 1M = 203.3 g/L, therefore 1.5 mM = 0.076 g/250 mlEDTA: 1M = 372.2 g/L, therefo

39、re 0.2 mM = 0.0186 g/250 mlDTT: 1M = 154.2 g/L, therefore 0.5 mM = 0.019 g/250 ml26% Glycerol (v/v) = 65 ml4.6 M NaCl4.6 M NaCl87.66 g/326 mlTBS (Tris Buffered Saline) pH 7.6-7.8:TBS (Tris Buffered Saline) pH 7.6-7.8:For 10 litresFor 10 litres:60.6 g TRIS HCl13.9 g TRIS base87.66 g NaCl10 litres Ult

40、ra pure water (H2O)TBS 0.025% Triton X-100:TBS 0.025% Triton X-100:For 1 litreFor 1 litre:250 l Triton X-100999.75 ml TBSpH 7.6-7.81.6% H2O2 (Hydrogen Peroxide) in TBS:1.6% H2O2 (Hydrogen Peroxide) in TBS:For 400 ml:For 400 ml:6.4 ml H2O2 (GPR = 30% w/w)393.6 ml TBSpH 7.6-7.8BS - Blocking serum in T

41、BS (100ml)BS - Blocking serum in TBS (100ml):NGS (10%) 10mlBSA (2%) 2mlTriton (0.4%) 4ml of 10% Triton10% NS (Normal Serum) with 1% BSA (Bovine Serum Albumin, Fraction 5) in10% NS (Normal Serum) with 1% BSA (Bovine Serum Albumin, Fraction 5) inTBS:TBS:For 1 ml:For 1 ml:100 l NS10 mg BSA900 l TBSpH 7

42、.6-7.8Primary antibody made up in TBS with 1% BSA:Primary antibody made up in TBS with 1% BSA:(Example is of primary antibody used at a dilution of 1:10)For 0.1 ml:For 0.1 ml:100 l Primary antibody10 mg BSA900 l TBSpH 7.6-7.8Secondary biotinylated antibody made up in TBS with 1% BSA:Secondary biotin

43、ylated antibody made up in TBS with 1% BSA:(Example is of secondary biotinylated antibody used at a dilution of 1:200)For 1 mlFor 1 ml:5 l Secondary biotinylated antibody995 l TBSpH 7.6-7.8ABC (Avidin-Biotin) complex in TBS:ABC (Avidin-Biotin) complex in TBS:(Example is of ABC complex, each part use

44、d at a dilution of 1:100)For 1 ml:For 1 ml:10 l Streptavidin10 l HRP (or AP)-Biotin980 l TBSpH 7.6-7.8Bicarbonate/carbonate coating buffer (100 mM):Bicarbonate/carbonate coating buffer (100 mM):3.03 g Na2CO3,6.0 g NaHCO3 (1 L distilled water)pH 9.6,PBS (500 ml):PBS (500 ml):1.16 g Na2HPO4,0.1 g KCl,0.1 g K3PO4,4 g NaCl(500 ml distilled water) pH 7.4