Heya guys, long time no see. In a blink of an eye, 13 weeks of SIP/MP have already past. Next week is our 3rd campus discussion and hope to see you all soon. By the way, please refer to Miss Chew's blog for updates regarding whether there is a blog quiz.
Today, I am going to share on one of two methods of pre-electrophoresis preparation steps that are required for my project. The gels that i will be running are 1D-Zymogram, 2D-gel and 2D-Zymogram and the pre-electrophoresis preparation is essential for running the gels mentioned above. To refresh your memory, Zymography is an electrophoresis technique that is used in the detection of protease activity under non-denaturing conditions. It is performed on a zymogram gel, which incorporates the use of a substrate that is copolymerized with polyacrylamide gel . Proteases that catalyze Gelatin , Caesin or Fibrin as a substrate will show up as clearings against a dark blue background after staining with Commassie brilliant blue. (Please read on previous post entry for more information regarding zymogram )
Wthout further ado, the two Pre-electrophoresis preparation steps are Bradford assay and TCA (Trichloroacetic acid) precipitation. In this post, the focus will be on Bradford assay.
Principle of Bradford assay: Bradford assay is a protein colormetric assay that will produces a colour change if proteins are present. The coomassie dye is originally red in colour. However in the presence of protein binding, it changes colour and stabalises into coomassie blue, resulting in an absorbance shift. This happens because of 2 bond to bond interactions taking place. The red form of commassie dye donates free proton to ionized groups on protein disrupting its conformation. This leads to hydrophobic heads of the proteins being exposed. The expose hydrophobic pockets on protein chain bind to non-polar region of the dye by van der waals force. Hence, this positions the positive amine groups closely to negative charge of the dye. Ionic interaction further strengthens the bond and ther is blue coomassie dye. Binding of the protein stabilises blue form of coomassie dye and the complex is measured for protein concentration by absorbance reading at 595nm. If no protein is bound to the dye, the cationic (unbound form) are green or red while binding stabalises the anionic (bound form) are blue in colour.
By using Bradford assay, the periplasmic protein concentration in the supernatant can be determined. The mass of periplasmic proteins remained at a constant at 10ug. By knowing the mass and protein concentration of the protein, the volume of protein sample to be loaded into the wells of the gel can be determined. This is because of the formula: Concentration (ug/ul) = Mass (ug) / Volume (ul). The volume can be found by manipulating the formula: Volume = Mass / Protein concentration (determined by Bradford assay).
Methods and Explanation
1. Warm up Bradford dye reagent to room temperature
It will not affect the sample at cold temperature and works optimally at room temperature
2. Pour Bradford dye reagent to plastic tray and cover with aluminium foil
Bradford reagent is light sensitive and cannot be exposed to light
3. Prepare the centrifuge tubes and mixed in the appopriate standards ( Milli Q + BSA)
Allows a calibration curve to be plotted
4. Prepare the sample in 5X dilutions
This ensures that there will be enough sample left after pipetting, hence need to prepare excess
5. Centrifuge standards and sample (short spin for 7 seconds)
To thoroughly mixed the milli Q and BSA/sample
6. Pipette 5ul of sample or standards in triplicates into each well using reverse pipetting (microtitre plate)
Ensures average readings can be taken after spectrophotometry for accurate results
7. Add 250uL bradford reagent into each well using multichannel pipette and reverse pipetting
Reverse pipetting to ensure exactly 250uL bradford is actually added and not more or less. It also prevents air bubbles forming
Allows binding of bradford to proteins for spectrophotometry
8. Remove air bubbles present using a pipette tip dipped with ethanol
Prevent air bubbles in samples, lead to inaccurate results
9. Cover microtitre plate with aluminium foil and incubate 30 minutes
Allow the reaction to occur at room temperature
10. Set up the spectrophotometer
To measure absorbance reading at 595 nm and to quantitate amount of proteins
The need to use BSA Standards: BSA standards are prepared at the concentration of 0, 0.1, 0.25, 0.5, 0.75mg/ml to obtain a linear range (standard curve). When the commassie blue dye binds to the protein, absorbance reading is read using spectrophotometer and absorbance reading is interpolated to the linear range of Bradford Assay. The protein concentration can thus be obtained.
That's all for now. Thanks you for reading my post and have an enjoyable next 7 weeks!
From: Benjamin Ma