Bradford lab report 2017.pdf

SBL100​ ​LABORATORY Name​ ​&​ ​Entry​ ​No.:​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​Sudhanshu​ ​Singh(2016TT10877) ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​Rahul​ ​Modpur(2016EE30503)

Group​ ​No.:​ ​4

Introduction: General​ ​introduction The​ ​Bradford​ ​protein​ ​assay​ ​is​ ​a​ ​simple​ ​calorimetric​ ​assay​ ​for​ ​measuring​ ​total​ ​protein concentration​ ​and​ ​is​ ​based​ ​on​ ​the​ ​Bradford​ ​dye​ ​binding​ ​method​ ​. Principle​ ​involved

The​ ​Bradford​ ​assay,​ ​a​ ​colorimetric​ ​protein​ ​assay,​ ​is​ ​based​ ​on​ ​an​ ​absorbance​ ​shift​ ​of​ ​the​ ​dye Coomassie​ ​Brilliant​ ​Blue​ ​G-250.​ ​Under​ ​acidic​ ​conditions​ ​the​ ​red​ ​form​ ​of​ ​the​ ​dye​ ​is​ ​converted​ ​into​ ​its bluer​ ​form,​ ​binding​ ​to​ ​the​ ​protein​ ​being​ ​assayed.

Other​ ​methods

Biuret​ ​protein​ ​assay BCA​ ​protein​ ​assay

Aim:​ ​To​ ​understand​ ​how​ ​to​ ​visualize​ ​and/or​ ​“measure”​ ​biomolecules​ ​–​ ​in​ ​this​ ​case​ ​“proteins”. Materials​ ​and​ ​method: Spectrophotometer​ ​,​ ​microcentrifuge​ ​,​ ​falcon​ ​tubes​ ​,​ ​amber​ ​tube​ ​,​ ​micropipette​ ​,​ ​Ashoka​ ​leaves​ ​, Bradford​ ​reagent​ ​,​ ​BSA(​Bovine​ ​serum​ ​albumin​)​ ​,​ ​PBS(Phosphate​ ​Buffered​ ​Saline)​ ​,​ ​PEB(Plant Extraction​ ​Buffer)​ ​,​ ​dye(​Coomassie​ ​Brilliant​ ​Blue​ ​G-250)​ ​.

● Take​ ​9​ ​microtubes​ ​and​ ​fill​ ​the​ ​first​ ​one​ ​with​ ​400​μ​L​ ​of​ ​PBS​ ​with​ ​the​ ​help​ ​of​ ​micropipette and​ ​then​ ​ ​keep​ ​on​ ​reducing​ ​10​μ​L​ ​with​ ​each​ ​successive​ ​tube. ● Add​ ​appropriate​ ​volume​ ​of​ ​BSA​ ​such​ ​that​ ​volume​ ​in​ ​each​ ​tube​ ​becomes​ ​400​ ​μ​L. ● Now​ ​fill​ ​each​ ​tube​ ​with​ ​1.6mL​ ​of​ ​Bradford​ ​reagent​ ​. ● Incubate​ ​the​ ​tubes​ ​in​ ​dark​ ​for​ ​10​ ​min​ ​. ● Calculate​ ​the​ ​OD(Optical​ ​Density​ ​)​ ​of​ ​each​ ​tube​ ​with​ ​the​ ​help​ ​of​ ​a​ ​spectrometer​ ​. ● Take​ ​a​ ​Ashoka​ ​leaf​ ​to​ ​extract​ ​protein​ ​with​ ​the​ ​help​ ​of​ ​PEB​ ​. ● Fill​ ​a​ ​tube​ ​with​ ​360​μ​L​ ​of​ ​PBS​ ​and​ ​then​ ​add​ ​40​μ​L​ ​of​ ​extract​ ​to​ ​it​ ​.Finally​ ​add​ ​1.6mL of​ ​Bradford​ ​reagent​ ​. ● Place​ ​the​ ​tube​ ​in​ ​microcentrifuge​ ​to​ ​separate​ ​the​ ​proteins​ ​from​ ​other​ ​compounds . ● After​ ​separation​ ​remove​ ​the​ ​protein​ ​from​ ​it​ ​and​ ​divide​ ​it​ ​into​ ​two​ ​parts​ ​. ● Measure​ ​the​ ​OD​ ​of​ ​one​ ​of​ ​the​ ​tubes​ ​. ● Divide​ ​the​ ​remaining​ ​tube​ ​in​ ​two​ ​equal​ ​proportions​ ​and​ ​label​ ​it​ ​as​ ​4°C​ ​and​ ​25°C​ ​. ● Store​ ​the​ ​two​ ​tubes​ ​at​ ​their​ ​respective​ ​temperatures​ ​. ● Measure​ ​the​ ​OD​ ​of​ ​both​ ​tubes​ ​after​ ​a​ ​week​ ​.

Observation​ ​and​ ​Results:

● The​ ​graph​ ​is​ ​fairly​ ​linear​ ​as​ ​expected​ ​from​ ​the​ ​Beer-Lambart​ ​law​ ​. where​ ​A​ ​corresponds​ ​to​ ​absorbance(OD) l​ ​corresponds​ ​to​ ​length c​ ​corresponds​ ​to​ ​concentration from​ ​the​ ​equation​ ​we​ ​can​ ​see​ ​that​ ​A​ ​is​ ​directly​ ​proportional​ ​to​ ​concentration​ ​. ● The​ ​protein​ ​degrades​ ​with​ ​time​ ​therefore​ ​the​ ​OD​ ​measured​ ​after​ ​a​ ​week was​ ​less​ ​compared​ ​to​ ​the​ ​original​ ​sample​ ​. ● But​ ​storing​ ​the​ ​protein​ ​at​ ​lower​ ​temperatures​ ​slows​ ​down​ ​the​ ​process​ ​of degradation​ ​.​ ​Therefore​ ​OD​ ​of​ ​4°C​ ​was​ ​more​ ​than​ ​25°C​ ​sample​ ​.