Western Blot Diary 20th Nov 2024

In the lab today we worked on the Bradford Assay and preparing our standards and pipetting onto a well plate to read the absorbance readings on a plate reader

  • Why is Bradford assay related to our dissertation?

    • we need to determine the concentration of the proteins

The Bradford assay is a widely used, colorimetric protein quantification method based on the binding of a dye, Coomassie Brilliant Blue G-250, to proteins. This assay is simple, fast, and effective for estimating the concentration of proteins in a solution.

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In this procedure we have to prepare the standards which we will place for the standard curve. To create the standards we used serial dilutions and created this table below. However we first created the stock solution of BSA.

Materials we used during the practical :

  1. Bovine Serum Albumin (BSA) powder

  2. An appropriate buffer = phosphate-buffered saline, PBS

  3. Micropipettes and tips.

  4. Volumetric flasks or tubes. Documented

BSA STOCK SOLUTION PROCEDURE:

  1. Calculate the Mass of BSA Required:

    • Desired concentration = 10 mg/mL.

    • Total volume = 100 mL.

  2. FORMULA TO CALCULATE BSA WEIGHT

    • Example: Mass of BSA required (mg) = Desired concentration (mg/mL)× Volume (mL)

    • Example: Mass of BSA = 10 mg/mL × 100 mL = 1000 mg OR  (1 g) BSA Mass

    • However. In our lab we did: MASS = 10 mg/mL x 2 mL

    • Therefore the mass we need in this practical was: = 20 mg of BSA powder

  3. Weigh the BSA:

    • We Used a precision balance to measure 0.02 g of BSA powder accurately which is the same as 20 mg of BSA powder.

  4. Dissolve the BSA:

    • We Added the weighed BSA to a container with approximately 80–90 mL of PBS buffer.

    • We Mixed thoroughly using a magnetic stirrer and by gentle shaking until the BSA is completely dissolved.

  5. Adjust the Final Volume:

    • We Transfer the solution to a 100 mL volumetric flask

    • Then Added remaining PBS buffer to bring the total volume to 100 mL on the volumetric flask.

  6. Mix Again:

    • Ensure the solution is homogeneous meaning evenly distributed throughout the liquid by stirring or gently inverting the flask.

    • If not homogenous in a Bradford assay, an uneven solution can result in incorrect protein concentration readings.

PREPARING STANDARD CURVE KNOWN CONCENTRATIONS

Once we prepared our stock solution it is time to prepare dilute our stock solutions into known concentraions for the standard curve

Use the following formula to calculate dilutions: C1​ x V1​ = C2​ x V2​

  • C1​ = 10mg/mL (stock concentration)

  • C2​ = Desired concentration (in mg/mL) (e.g 0.05 mg/mL)

  • V2 = 1 mL (final volume)

  • We have to Solve for V1​:

Here was our example dilution table for a stock solution of 10 mg/mL and 1 mL final volume

IMPORTANT

  1. We multiply (C2 x V2) then we divide by the final volume (V2) to find V1

  2. C1 is implied through the table as 10 mg/mL

  3. To find diluent volume we need to do a calculation

  4. Therefore we will need to do FINAL VOLUME - STOCK VOLUME = DILUENT VOLUME

  5. The diluent volume, this will be the remaining PBS buffer to dilute the BSA stock volume

Final Concentration (mg/mL) (C2)

Stock Volume (V1) (µL)

Diluent Volume (µL)

Final Volume (µL) (V2)

0.05

(0.05×1000)/10

= 5 µL

1000 − 5

= 995 (µL)

1000

0.1

(0.1×1000)/10

= 10 µL

1000−10

= 990 (µL)

1000

0.2

(0.2×1000)/10

= 20 µL

1000−20

= 980 (µL)

1000

0.3

(0.3×1000)/10

= 30 µL

1000−30

= 970 (µL)

1000

0.4

(0.4×1000)/10

= 40 µL

1000−40

= 960 (µL)

1000

0.5

(0.5×1000)/10

= 50 µL

1000−50

= 950 (µL)

1000

0.6

(0.6×1000)/10

= 60 µL

1000−60

= 940 (µL)

1000

0.8

(0.8×1000)/10

= 80 µL

1000−80

= 920 (µL)

1000

1.0

(1.0×1000)/10

= 100 µL

1000−100

= 900 (µL)

1000

1.2

(1.2×1000)/10

= 120 µL

1000−120

= 880 (µL)

1000

1.4

(1.4×1000)/10

= 140 µL

1000−140

= 860 (µL)

1000

MICROPLATE PROCEDURE

The next step once we finished with our standards was to pipette them onto a 96 well-plate.

Bradford Protein Assay

  1. We pipetted 10 µL of EACH STANDARD in triplicate onto the well plate

  2. Then added 200 µL of Bradford Plus Assay Reagent into the well plate with the standard

Unknown Protein Samples

  • These unknown proteins concentrations was prepared by Dr. Caprifico

These proteins were kept in freezer at -20 degrees to ensure that the proteins do not denatured because it wont be able to react with the Bradford assay to give the colimetirc change.

Pipette Technique

We learned the importance of using the pipette and how to hold it correctly

  • We made sure every sample was dispensed in the corner of the well to avoid droplets on the side wall

  • This is to make sure all the sample can mix and react with the reagenets and able to give a clear reading on the plate reader at the final stage

  • Avoiding cross contamination with the tips of the pipettes.

  • Any cross contamination will relsut in coulr chamnge happening too early which. e.g. contaminating the Bradford assay can result in early colour change which can give poor readings in the plate reader in the end stage.

  • Also poor contamination control can cause wasategfe or reagent whihc costs more money to buy for the staff at the university

PLATE READER FINAL STAGE

Finally after completing the standards and our proteins which have all been reacting with the Bradford assay and 10 mins incubation, we can use a plate reader to gather the absorbance data and create a standard curve on Excel.

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EXCEL RESULTS

Our Standard curve Results screenshot Our Protein Results Screenshot