Department Of Biochemistry And Microbiology Biology Essay
Adenosine triphosphate is a mononucleotide with an adenine base and a ribose sugar to which three phosphate groups are linked. The covalent bond between the 2nd and the 3rd phosphate group is unstable and easy broken by hydrolysis, thereby let go ofing energy ( Kent, 2000 ) . ATP can therefore shop energy so as to be used to drive endergonic reactions through a procedure of phosphorylation. Phosphorylation refers to the transportation of energy with the add-on of a phosphate group from ATP to an endergonic reaction leting for the reaction to be driven frontward. ATP is hence indispensable for life as it provides the much needed energy to drive a life system ( Alters, 2000 ) .
There are assorted ways to quantify ATP, one of which is through soaking up spectrometry by ultraviolet visible radiation. The optical density of visible radiation, at a given wavelength, by a substance with chromophore belongingss consequences in an exponential bead in light strength ( Wilson and Walker, 2010 ) . With the cognition of the substances ‘ molar extinction coefficient or the step of how strongly the substances absorbs visible radiation at a given wavelength, the concentration of the substance can be determined with the usage of the Beer-Lamberts Law: , where A refers to absorbance at the given wavelength, ? refers to the molar extinction coefficient, c the concentration of the sample and l the distance visible radiation travelled through the sample ( Bansal, 2003 ) . Purine bases such as A, a cardinal constituent of ATP, absorb UV visible radiation between 260 and 275nm leting for their quantification by this method.An extra method of quantifying ATP can be carried out indirectly by an assay process for pentose sugar, viz. ribose.
As discussed earlier ATP contains one ribose molecule leting for the premise that 1 mol of ribose will account for 1 mol of ATP within a sample of pure ATP. Ribose in an acidic solution will ensue in the formation of furfural which in bend reacts with orcinol, bring forthing a green coloring material. The strength of the green coloring material can hence be used to spectrophotometrically quantify ribose at 660nm and indirectly ATP, sing no extra pentose sugar contaminates are present ( Nigam, 2007 ) .Many applications require the quantification of protein, frequently in a high throughput mode as can be expected within assorted research labs. One such method involves the usage of the bicinchoninic acid ( BCA ) protein assay developed by Smith et Al. The assay depends on the transition of to under alkaline conditions which in bend reacts with BCA ensuing in an intense purple coloring material, which can be quantified through spectrometry techniques at an optical density of 562nm.
The production of in the check is a map of the protein concentration and the incubation clip, leting for the protein quantification ( Walker, 1994 ) . In add-on, fluorimetric protein quantification can be carried out with the assistance of a reactive compound ; fluorescamine. Fluorescamine is a really sensitive fluorogenic reagent which reacts with primary amides to organize fluorescent pyrrolinones.
This consequences in a green-yellow fluorescence which can be quantified as a map of protein amide groups ( Rost, 1995 ) , such as the terminal protein amide groups and ?-amino group of lysine ( Rosenthal and Wright, 2005 ) .Other general techniques frequently used within a biochemical research lab are centrifugation techniques. In this instance differential centrifugation is the focal point. The procedure is based upon the differing deposit rates of biological atoms as a map of differing size and denseness. For illustration, this allows for the division of petroleum homogenates into assorted fractions of cell organs, membrane cysts and other structural fragments by agencies of a bit-by-bit addition in the applied centrifugal field and centrifugation times ( Wilson and Walker, 2010 ) . One of the authoritative illustrations is the bit-by-bit separation of liver homogenate to insulate liver chondriosome, whereby the effectivity of the technique can be analyzed through a succinate dehydrogenase ( SDH ) assay.
Succinate dehydrogenase exists ubiquitously within chondriosomes in all aerophilic tissue whereby the enzyme catalyses the dislocation of succinate to fumarate organizing FADHa‚‚ in the procedure. The dislocation of succinate can be used as a mitochondrial index by add-on of blue/purple oxidation-reduction dye viz. dichlorophenol indophenols ( DCPIP ) . Blue DCPIP acts as a H acceptor from FADHa‚‚ ( i.e. merchandise of the SDH enzyme ) ensuing in a loss of coloring material during the procedure, which can be straight quantified spectrophotometrically at an optical density of 600nm and thereby related to mitochondrial concentration ( Nigam, 2007 ) .The purpose of the experiment is to analyse assorted techniques to find ATP and protein concentrations of unknown ATP samples A.
In add-on, centrifugation techniques with a poulet liver homogenate as a theoretical account will be addressed, making fractions which are to be assayed for mitochondrial activity.
1. Quantification of ATP utilizing Absorption by Ultraviolet LightThe quantification ATP of an unknown ATP sample A ( group 1 ) was carried out by agencies of ultraviolet soaking up spectrometry. The unknown sample of 0.45g was diluted to do a stock solution of 10mg/ml and for farther diluted as required for subsequent optical density spectrometry processs ( e.g. 1:10 or 1:100 dilution ) . The optical density spectrum from 220 to 310nm of the unknown ATP sample was performed and the content of the ATP was determined from the optical denseness at 295nm.
The molar extinction coefficient of ATP at pH 7 at 295nm was taken to be.2. Quantification of ATP utilizing the Ribose Content2ml of the unknown ATP sample A solution was assorted with 2ml of 1 % orcinol which was dissolved in 0.
1 % FeCla‚? in concentrated HCl. The mixture was prepared in trial tubings and sealed with cotton spiles. The mixtures were so heated for 30min in a boiling H2O bath. The mixture was so cooled and so diluted to 4ml. The optical densenesss of the samples were so observed at 660nm. A standard curve was besides created runing from 0.01 – 0.
1 mg/ml whereby the unknown sample was substituted for known concentrations of ribose. The whole process for finding the ribose content was carried out in triplicate. Mention to Postpone 3.1 for assay process within the appendix.
Biochemical Techniques – Protein Determination Methods
1. VIS Microtiter Plate Quantification of Protein utilizing the Bicichonic Acid ( BCA ) assayA BCA check process was carried out to find the protein content of unknown ATP sample A with the usage of a 96-well VIS microtiter home base reader. A five point criterion curve of known BSA was created ( 0 -2 mg/ml ) . Mention below to Postpone 1.1 for BCA assay process. Mention to Postpone 3.
2 of appendix for the assay process.Table 1.1: BCA assay for the finding of unknown ATP sample A within Bovine Serum Albumin ( BSA ) criterion curveReagentVolume ( µl )Sample/BSA10Reagent A and Reagent B* ( 50:1 )200Incubated at 37 & A ; deg ; C for 30min. Optical density read at A540nm*Refer to appendix for components of reagent A and B2. Fluorimetric Quantitation of Protein utilizing the Reactive Compound FluorescamineA five point criterion curve of BSA was constructed runing from 0 – 500µg/ml, diluted in phosphate buffered saline ( PBS ) at pH 7.
4 to find the unknown ATP sample A protein concentration. 150µl aliquots were so transferred to a black 96-well microtiter home base followed with the add-on of 50µl of 3mg/ml fluorescamine dissolved in propanone. The home base was so shaken for 1min followed by fluorescence finding with a 355nm excitement filter and a 460nm emanation filter. All samples were carried out in triplicate. Mention to Postpone 3.3 of appendix for assay process.
Mitochondrial enzymatic activity was determined whereby 50g of poulet liver tissue was obtained and minced before being diluted in 10ml of sucrose isolation medium ( 300mmol/l saccharose: 0.5mmol/ EDTA at pH 7.
4 ) per gm of tissue. The sample was so homogenized with a co-axial homogenizer. The howitzer and stamp was so fixed to a drill ensuing in comparative howitzer motion to the stamp, revolving at 2400rpm for 8-10 complete shots. Procedures were performed at 4 & A ; deg ; C. Centrifugation of the suspension was so carried out as observed in Figure 1.1. However, the last centrifugation measure was altered to be 40000xg for 120min as opposed to 100000xg for 120min due to the rotor of ultracentrifuge merely being rated to 40000xg.*Note aliquots of each fraction ( H1, S1, S2, S3, P1, P2, and P3 ) were retainedFig.
1.1: Differential centrifugation process carried out on poulet liver homogenateA succinate dehydrogenase ( SDH ) check was performed on the homogenate, supernatant and pellet samples obtained during differential centrifugation of the liver sample. Assay medium was created as can be seen in the appendix ( Table 3.4 ) to be used in the SDH check.
For each sample 2.8ml of the check medium was pre-warmed to 37 & A ; deg ; C followed by the add-on of 0.2ml of the dilute enzyme within the homogenate, supernatant or pellet samples and the optical density was determined at 600nm. This was taken to be clip zero ( ta‚ˆ ) .
The subsequent optical density values were recorded at 1min clip intervals for a sum of 5min. A advancement curve was so plotted for each fraction to find the alteration in optical density over clip. The sucrose isolation media was used as a space.
1. Quantification of ATP utilizing Absorption by Ultraviolet Light:Absorption of ultraviolet visible radiation by ATP is a map of the A group. The given molar extinction coefficient of ATP at pH 7 at 295nm is.
The optical denseness of sample A was determined to be 0.664nm at an ABS of 295nm:Therefore the Beer-Lamberts jurisprudence provinces:Where A is the optical density, ? is the molar extinction coefficient, degree Celsius is the concentration of the sample and is the way length ( i.e. distance travelled by the visible radiation through the cuvette ) .Therefore:Therefore sample A containedof ATP, nevertheless the sample pH was higher and did non correlate to the pH of 7 that allowed for the premise of a molar extinction coefficient of.Fig.
2.1: The optical density spectrum of unknown ATP sample A ranging from 210nm to 290nm. An optical density extremum of 260nm was observed that correlative to, and hence confirms the presence of ATP.Fig 2.2: Standard curve for the quantification of ATP utilizing the ribose content. It was assumed that 1mol of ribose amounted to 1mol of ATP.
The equation for the best fit line: with a additive arrested development of 0.901. The unknown ATP content of sample A was determined to be 2.425 mg/ml. Note a 1:100 dilution factor was used.Calculations:* 1:100 dilution factorIn respects to the five point criterion curve ( fig.2.
2 ) , the ribose standard concentrations of 0.05 mg/ml was omitted. This was due to spectrometry mistakes as the instruments showed changing absorbencies in the higher concentrations values which did non match to expected consequences after multiple replicates. It is believed this was due to instrument harm during redevelopments to the Nelson Mandela Metropolitan University ( NMMU ) biochemistry section.Fig 2.3: Standard curve for the VIS microtiter home base protein quantification of the unknown sample A, utilizing the bicinchonic acid ( BCA ) assay. The equation for the best fit line: with a additive arrested development of 0.
998. The unknown protein content of sample A was hence determined to be 1.226mg/ml. Note a 1:10 dilution factor was used.Fig 2.4: Standard curve for the quantification of unknown protein of sample A by agencies of the fluorescamine check. The equation for the best fit line: with a additive arrested development of 0.977.
The unknown protein content of sample A was hence determined to be 3.191mg/ml.In respects to fig2.4 the concluding point on the curve that amounted to a concentration of 500µg/ml, was omitted so as to increase the correlativity coefficient closer to 1.0.
This was due to high criterion divergences and emanation values which did non match to the concentration.Table 2.1: The ATP and Protein concentration and per centum of unknown sample AMethodConcentrationPercentage
ATP ( mg/ml )
Protein ( mg/ml )
ATP ( % )
Protein ( % )
UV Quantification by extinction coefficient0.0219
Quantification by Ribose Content2.425
BCA Protein Assay
26Fluorescamine Protein Assay
31.91High disagreements between ATP and protein quantification was observed between the given techniques, as big per centum fluctuations were apparent.Calculations ( e.g. UV Quantification by extinction coefficient ) :*100Fig 2.
5: Succinate dehydrogenase ( SDH ) activity assay bespeaking the alteration in optical density @ 600nm as a map of mitochondrial concentration within the given fractions. The undermentioned fractions were assayed: the homogenate ( H ) , pellet 1, 2 and 3 ( P1, P2, and P3 ) and the supernatants 1, 2, and 3 ( S1, S2, and S3 ) .The greatest lessening in optical density ( fig 2.5 ) was observed to be the homogenate fraction ( H ) , followed by the first pellet ( P1 ) .
P1 was obtained after the centrifugation measure at 3000xg for 20min. The subsequent fractions did non give high lessenings in optical density as compared to the H and P1 fractions and hence did non demo important mitochondrial activity.Fig 2.6: Bar graph representation of the succinate dehydrogenase ( SDH ) activity assay as seen in fig 2.5. Parallel barss indicate the alteration in optical density @ 600nm over the initial first minute of the reaction.
Determination of succinate dehydrogenase activity ( e.g. Fraction H ) :Ten -1X-1As observed in fig 2.5 the highest grade of activity was observed for the homogenate fraction ( H ) followed by the first pellet ( P1 ) .
The subsequent fractions did non demo high mitochondrial activity in retrospect and hence were non important.
The quantification of ATP of the unknown sample A by ultraviolet visible radiation as a map of Beer-Lamberts Law showed that 0.219 % of the sample comprised of ATP. This was assumed that the conditions for soaking up of ATP was changeless and related to old surveies where the molar extinction coefficient was determined at pH 7.
The unknown sample A did non nevertheless, have a pH of 7 but one that was well higher which would take to possible inaccuracies in the quantification. The neutering of the samples environment due to pH incompatibilities consequences in protonation/deprotonation of the sample and hence affects the distribution of negatrons within the chromophore. This will correlate to differences between optical density spectrums at changing pH values ( Sheehan, 2009 ) . However, when taking the ascertained optical density spectrum of the unknown sample A into history ( fig.2.
1 ) , there were no important displacements in the spectrum. This would bespeak no true changes the construction of the sample occurred due to the incorrect pH value and sick consequence on the ascertained ATP concentration calculated. The method for pH quantification assumed that the molar extinction coefficient for ATP was at a wavelength of 295nm, nevertheless this is wrong. A molar extinction coefficient of this value for ATP correlates to a wavelength of 259nm and non one of 295nm therefore quantification of the sample was measured falsely and can non be considered accurate ( Gerstein, 2004 ) . The soaking up spectrum ( fig.2.
1. ) for ATP strengthens this decision as an optical density extremum was observed at 260nm ( i.e. really near to 259nm ) and non near 295nm. Due to these inaccuracies the consequences obtained for the unknown ATP concentration can non be consider valid and therefore finding relies on the ATP quantification by a map of ribose content and the orcinol check.The orcinol check for ribose content yielded consequences that indicated that 24.25 % of the unknown sample A comprised of ATP. However, these consequences in add-on can non be considered accurate.
The spectrophotometery instrumentality used produced changing consequences chiefly at the higher optical density values at a wavelength of 660nm. It was determined that this was due to decalibration and/or harm to the instrumentality during redevelopments to the research lab where the instruments were stored. This resulted in the remotion of the higher concentration points of the standard curve to obtain a respectable correlativity coefficient ( fig.2.2. ) . For an accurate consequence in this instance the concentration of the unknown sample is required to fall between the first three concentration/absorbance values of the standard curve.
However, the true sum of harm sustained by the spectrophotometery instrumentality can non be to the full assessed and hence the consequences obtained should non be considered.The quantification of ATP of unknown sample Angstrom by both methods viz. ; quantification of ATP by soaking up of UV visible radiation and the quantification of ATP by the finding of the ribose content were non accurate. Therefore, alteration of the methods should be carried out whereby the pH is more strictly controlled and quantification should be carried out at the right wavelength ( i.e. 259nm ) in the instance of the first method. In the instance of the 2nd method, the truth of the instruments used should be verified prior to utilize.
There are nevertheless, other methods to find the ATP content of a sample more accurately, one of which is high public presentation liquid chromatography ( HPLC ) . Manfredi et Al. in their measurings of ATP in mammalian cells optimized a HPLC protocol for the quantification of ATP, ADP and AMP. With the usage of ion-pair reverse-phase HPLC system with a phosphate-buffered acetonitrile gradient nomadic stage the ATP of the unknown sample A can be quantified ( Manfedi et al. , 2002 ) . This method will be more effectual as contaminates would non interact and interfere with the ATP due to changing dissociation coefficients leting for merely ATP quantification.Protein finding by the bicinchonic acid check has been determined to be sensitive to 0.
5-10µg protein/ml when performed as a microassay, and is in most instances chosen over the Lowry assay method as it is more tolerable to interfering compounds ( Walker, 1994 ) . By and large the optical density used for the BCA check is 562nm nevertheless, in this instance, an optical density of 540nm was used. In respects to filtrate based home base readers a wavelength scope of 540 to 590nm can be used when executing the reaction without a big grade of sensitiveness loss ( Burgess and Deutscher, 2009 ) .
Therefore, any inaccuracies that may hold occurred due to the chosen wavelength do non hold to be considered in this instance. From this method it was determined that 12.26 % of the unknown ATP sample A contained protein, nevertheless an absolute concentration of protein can non be determined. This is due to the fact that the BCA check is dependent on the amino acerb composing of the protein, and hence disagreements will originate when utilizing a bovine serum albumin criterion curve ( BSA ) . That is to state the BSA amino acid/peptide composing will differ somewhat to that of the unknown protein sample ( Walker, 1994 ) .
Fluorescamine check for protein finding in comparing to the BCA assay process is well more sensitive. Fluorescamine allows for the quantification of proteins at degrees every bit low as 25-50pmoles ( Lawrence and Frei, 2000 ) . It was determined from the fluorescamine assay that 31.91 % of the unknown sample A comprised of protein which is over twice that determined by the BCA check. The sum of protein nowadays was determined through the 1:100 dilution of the unknown ATP sample A as fluorescamine has a greater sensitiveness at lower concentrations and this dilution yielded consequences with the lowest standard divergence. The fluorescamine check was carried out at a pH of 7.
4 which is non ideal for maximal sensitiveness as the low pH can strongly act upon the fluorescamine strength. Maximum sensitiveness occurs at a pH of 9 ( Keyes, 2000 ) . For best consequences the method should be altered to account for the alteration in pH and compared to the old fluorescamine assay consequences on the unknown sample A. This will let for a comparing that will demo whether or non the ascertained unknown sample A protein concentration was so altered by the low pH. At this phase nevertheless, it can be assumed that the protein content of the unknown sample A was in the part of about ±20 % when sing both BCA and the fluorescamine check consequences.In decision, the finding of the ATP content of the unknown sample A by the two stipulated methods was unsuccessful.
This was due to incorrect optical density values in the instance of ultraviolet quantification and faulty spectrophotometery instruments. In the instance of the protein finding of unknown sample A by the two stipulated methods disagreements between the methods arose which did n’t let for absolute protein quantification. However, a unsmooth estimation could be determined but would be unequal for most subsequent applications with the sample.
Differential centrifugation of liver homogenates for mitochondrial isolation by and large follows a really similar process, where the homogenate is foremost centrifuged at a low centrifugal force to take cell dust and karyon ( 700xg ) . The centrifugation of the subsequent supernatant is carried out at a much higher centrifugal force ( e.g. 20000g ) to divide most of the chondriosome from the fraction. This would ensue in the 2nd pellet holding the highest mitochondrial activity in comparing to any possible fractional process stairss carried out ( Ghosal and Srivastava, 2009 ) .
In respects to the method used in this experiment the first centrifugation measure was performed at a high centrifugal force ensuing in most of the chondriosome nowadays in the first pellet as opposed to the 2nd pellet in the general isolation techniques. This would account for the index enzyme, succinate dehydrogenases ‘ high observed activity in pellet 1. The homogenate besides showed really high mitochondrial activity as to be expected as it would incorporate the highest concentration of chondriosome. However, in relation to pellet 1 the homogenate should non hold every bit high a degree of mitochondrial pureness due to the presence of the cell dust and karyon which would interact and interfere with enzymatic reactions. The low degrees of succinate dehydrogenase activity in the subsequent fractions after pellet 1 would be an indicant of mitochondrial cross taint during the stepwise fractional process process. That is to state residuary chondriosome remained within the supernatant of pellet 1. This would be due to the comparatively low centrifugal force applied to the homogenate to make the mitochondrial pellet leting for residuary chondriosome to stay in the supernatant.For a higher isolation output of mitochondrial isopycnic centrifugation should hold been carried out leting for a purer mitochondrial merchandise.
The fraction enriched in chondriosome ( i.e. pellet 1 ) would be subjected to resuspension in a 20 % saccharose solution and so placed in a centrifugation tubing which contains a saccharose gradient. The gradient would be required to be 20 % sucrose at the top of the centrifugation tubing and 50 % sucrose at the underside. High velocity centrifugation will therefore separate the chondriosome from the lysosomes and peroxisomes as a map of denseness giving higher declaration ( Vignais and Vignais, 2010 ) .
In decision it was observed that mitochondrial isolation from a poulet liver sample occurs in the first or 2nd fraction of bit-by-bit differential centrifugation, depending on protocol fluctuations. This method of isolation is sufficient sing a low degree of pureness is acceptable for the coveted application.
Table 3.1: Assay process for finding the ribose content of unknown ATP sample 1ReagentSpace12345Unknown 1:100Ribose venereal disease conc.00.006250.01250.
Ribose venereal disease. *00.250.
511.520Ha‚‚O21.751.510.500ATP ( Unknown )
2Orcinol Reagent2222222Cover tubings, heat for 30min in boiling H2O bath, cool, dilute to 4mlA660nm ( 1 )0.0000.1230.2270.
290A660nm ( 2 )0.0000.1280.1830.3810.
4230.3170.380A660nm ( 3 )0.
0000.0030.0430.0160.0500.0430.047* Ribose Std. = 0.
05mg/mTable 3.2: The process for finding of the protein content of unknown sample A with usage of bicinchonic acid assayReagentSpace12345UnknownUnknown 1:10Unknown 1:100BSA Conc.0.000.501.001.
BSA std* ( µl )0.002.505.006.507.
5010.000.000.000.00BSA ( Unknown ) ( µl )
002.00Reagent A and B ( µl )200200200200200200200200200Incubate for 30min at 37 & A ; deg ; CA540nm ( 1 )0.0000.2340.4630.6120.8070.
8430.6030.0490.001A540nm ( 2 )0.0000.
0510.028A540nm ( 3 )0.0000.2440.4900.
014* BSA Std. = 2mg/mlTable 3.3: Assay process for finding of the protein content of bovine serum albumen ( BSA ) with usage of reactive compound fluorescamineReagentSpace12345UnknownUnknown 1:10Unknown 1:100BSA Conc. ( µg/ml )0100200300400500
BSA std* ( µl )0306090120150000PBS ( µl )1801209060300101010BSA ( Unknown ) ( µl )000000150150150Fluorescamine ( µl )505050505050505050Plate shaken for 1min and read at excitement of 355nm and an emanation of 460nmEX355nm/Em460nm ( 1 )0.
357822.157970.5571222.257189.05794.557EX355nm/Em460nm ( 2 )0.000199.157474.257777.5571382.257937.9571250.257186.55792.757EX355nm/Em460nm ( 3 )0.000150.857474.957905.4571401.257796.8572441.257164.75784.357Average0.000172.957498.723845.7901201.890901.7901637.923180.12390.557Standard Deviation0.00024.41041.77364.379328.99692.325695.84813.3675.444* BSA Std. = 500µg/mlTable 3.4: Components of the assay medium required for the succinate dehydrogenase ( SHD ) assayCompoundConcentrationSuccinate10mMDCPIP2.0mg/mlPhosphate buffer, pH 7.410mMKCN2mMCaCla‚‚10mMAlbumin0.5 mg/mlTable 3.5: Succinate dehydrogenase activity assay on the given liver fractions
FractionTime ( T )
02.9862.2901.3942.9021.4661.5650.80112.6442.0441.3902.8921.4291.5530.75322.5522.0221.3942.8011.4201.5450.72532.4402.0101.3992.8221.4181.5340.70942.1001.9831.4022.7581.4301.560.70251.3851.9571.4032.7551.4321.5640.697Change in Abs/min0.3420.2460.0040.0100.0370.0120.048*Note that the concentrated fraction samples were used for all activity checks and that optical density was measured at 600nm.
Diluting unknown sample A:Orcinol 1 % w/v, FeCla‚‚ 0.1 % w/v ( 600ml ) :Dilution of 100mg/ml ribose stock solution ( 300ml ) :Therefore 0.15ml stock solution to 299.85ml of dHa‚‚OReagent A ( 40ml ) :1.00 % : 0.4g BCA salt2.00 % : 0.8g Sodium carbonate0.10 % : 0.04g Sodium tartrate0.40 % : 0.16g Sodium hydrated oxide0.95 % : 0.38g Sodium hydrogen carbonateE.g. ( 1.00 % BCA salt )Reagent B ( 10ml ) :4.00 % Cu sulfateSucrose isolation media ( 4L ) :Sucrose ( 300mmol/L ) :Therefore:EDTA ( 0.5mmol/L ) :Therefore:Assay Medium ( 600ml ) :10mM Succinate – 708.5mg2.0mg/ml DCPIP – 1200mg10mM Phosphate Buffer, pH 7.4 – 1.014g ( 650ml made )2mM KCN – 78.1mg10mM CaCla‚‚ – 882.1mg0.5mg/ml Albumin – 300mgE.g ( 10mM Succinate )PBS – 10mmol PO4 and 0.9 % NaCl:POa‚„ : 0.0626gNaCl: 0.3607gDistilled Water: up to 40ml