In bio labs, it’s often necessary to prepare common buffers for various experiments. I’ve decided to create this post as a memo for myself as a handy reference. Over time, I’ll be updating this post with buffer recipes (often available on the Internet somewhere) with helpful tips and tricks for their preparation.
Gelatin Zymography is a method to detect the activity or gelatinize enzymes such as, in our particular interest, matrix metalloproteinases (MMP-2). The enzymes digest the gelatin embedded in the polyacrylamide gel, and the areas of degradation shows up as clear bands against a dark background.
Washing Buffer (Renature Buffer)
|Final Concentration||For 250 mL||For 1 L|
|2.5% Triton X-100||6.25 mL of 100%||25 mL of 100%|
|50 mM Tris-HCl, pH 7.5||12.5 mL of 1 M stock||50 mL of 1 M stock|
|5 mM CaCl2||625 uL of 2 M stock||2.5 mL of 2 M stock|
|1 uM ZnCl2||2.5 uL of 0.1 M stock||10 uL of 0.1 M stock|
For each reagents above, you can prepare some stock solutions for future use.
|Stock Reagent||How to Prepare|
|CaCl2 (2 M stock)||Add 11.1 g CaCl2 in 50 mL dH2O.|
|ZnCl2 (0.1 M stock)||Add 0.681 g ZnCl2 in 50 mL dH2O.|
|Tris base solution (1 M stock)||Add 30.275 g Tris base in 200 mL dH2O. Add HCl until pH reaches 7.5.|
Preparing the 1M Tris-HCl requires a little bit of effort. Make sure you open your HCl container in a chemical fume hood if you are using 36%~ HCl. After dissolving 30.275g Tris base in 200 mL dH2O, add roughly 16-17 mL of HCl until the pH of the solution reaches 7.5. You can sterilize the solution in an autoclave, but for gelatin zymography it’s okay not to.
The trick to preparing the washing buffer is patience. Patience is key because Triton X-100 is a very viscous liquid. It will immediately clump at the bottom of your bottle. Put in a stirring bar and leave it there until the solution eventually dissolves.
In western blot, we use antibodiesto identify proteins that have been separated based on size by SDS-PAGE. A detailed description of the immunoassay after membrane transfer can be found in my previous blog post. Since this post is more focused on buffer recipes, I will not go over too much detail in the technical and mechanical side of western blot.
10X Tris-Buffered Saline (TBS) Stock
TBS is a wash buffer for western blot. The solution concentration of this buffer is 200 mM Tris and 1500 mM NaCl. Add the following to 900 mL Milli-Q water for 1 L of buffer:
- 24 g of Tris Base
- 88 g of NaCl
- Adjust pH to 7.6 with 12 N HCl
- Add more Milli-Q water to 1 L.
1X TBS-T Wash Buffer
The final T here refers to Tween 20. It is a commonly used detergent that can help preventing non-specific antibody binding. The solution concentration of this buffer is 20 mM Tris, 150 mM NaCl, and 0.1% Tween 20.
To prepare this, add the following to 900 mL Milli-Q water for 1 L of buffer:
- 10X TBS Stock (see above)
- 1 mL of Tween 20 detergent
BSA Blocking Buffer
BSA = Bovine serum albumin.
This blocking buffer is used for saturating excessive protein-binding sites on membranes in Western blotting and microplates in ELISA.
For most experiments, a concentration of 1-3% BSA (Bovine Serum Albumin) is typically adequate. However, in our lab, we prefer to use 5% BSA.
This is a very easy buffer to prepare, and we usually prepare them as we need them. To prepare a 10 mL solution of a 5% BSA blocking buffer, simply dissolve 0.5 gram of BSA in 10 mL of TBS-T.
Our lab uses Corning Collagen I from rat tail. This collagen product can be used as a thin coating for cell attachment. The following protocol is adapted from Corning’s certificate of analysis.
- Prepare a collagen working solution: dilute the stock collagen solution to 50 ug/mL with 0.02 N acetic acid.
- Add enough working solution to coat dishes at 5 ug/cm2.
- For 60 mm dishes, 2-3 mL should be sufficient.
- Incubate at room temperature for one hour.
- Aspirate the remaining solution.
- Rinse with PBS twice to remove acid.
- Use immediately or store in 2-8ºC for maximum one week under sterile condition.
Preparing 0.02 N acetic acid can be a pain depending on your lab. Open your glacial acetic acid in a chemical fume hood. I usually make 40 mL each time, as needed. Refer to the following:
- Glacial acetic acid at a 99.7% is roughly 17.4 N.
- To prepare a 0.02 N solution, slowly add 0.046 mL of your stock solution to 10 mL deionized water.
- Then, adjust to final volume of 40 mL with more deionized water.
Our lab had bought the 4 L glacial acetic acid from Sigma. It would be a pain to take out 0.046 mL from a 4 L glass bottle. I take a tiny bit of aliquot from the stock and use the aliquot as needed.
Mammalian Cell Culture
Mammalian cell culture is an important tool for research, clinical, and pharmaceutical applications. Cells isolated from animal tissues can be expanded in culture using various specialized media, such as DMEM, RPMI, and EBM-2, to study cell biology and disease or used for the production of antibodies, proteins, and vaccines. Immortalized mammalian cell lines can be grown in vitro for prolonged periods and are commonly used as simple models for complex biology. Cell culture medium are usual basal medium upon purchase, meaning that you have to add cell growth supplements to them. Fetal bovine serum (FBS) at 10% is the most commonly used cell growth supplementation.
Dulbecco’s Modified Eagle Medium
Dulbecco’s Modified Eagle Medium (DMEM) is a widely used basal medium for various mammalian cells, and we use it in our lab. It has four times the amino acid and vitamin concentration of original Eagle’s Minimal Essential Medium (MEM), and offers options for different glucose levels and includes or excludes sodium pyruvate. DMEM maintains pH with a sodium bicarbonate buffer under 5–10% CO2. FBS is needed.
RPMI 1640 Medium
RPMI 1640 Medium is distinct due to glutathione and high vitamin levels. It includes biotin, vitamin B12, and Para-aminobenzoic acid (PABA or Vitamin Bx), absent in MEM and DMEM. High concentrations of inositol and choline are also present. RPMI 1640 Medium lacks proteins, lipids, and growth factors, and therefore also needing 10% Fetal Bovine Serum (FBS). It relies on a 2.0 g/L sodium bicarbonate buffer, maintaining pH under 5–10% CO2.