What are stain-free gels?
Stain-Free gel technology enables detection of protein bands in gels and on transfer membranes without using colorimetric or fluorescent stains. Stain-Free gels (Bio-Rad) contain a trihalocompound within the gel matrix that produces a fluorescent product when covalently crosslinked to protein tryptophan residues.
What is a stain-free blot?
Stain-Free technology allows scientists to visualize strong total protein signals with low background on their gels and blots that would otherwise not be possible without additional staining steps thus, improving overall dynamic range without the need to perform extra staining and destaining steps.
Can you stain a stain free gel?
With the fluorophore covalently bound to the protein molecules, they can be imaged repeatedly on a gel or membrane after transfer, without additional staining and destaining steps.
How do I make SDS PAGE gel?
- Prepare the separation gel (10%).
- Pour gel, leaving ∼2 cm below the bottom of the comb for the stacking gel.
- Layer the top of the gel with isopropanol.
- Remove the isopropanol and wash out the remaining traces of isopropanol with distilled water.
- Prepare the stacking gel (4%).
Why is SDS used in western blotting?
SDS is generally used as a buffer (as well as in the gel) in order to give all proteins present a uniform negative charge, since proteins can be positively, negatively, or neutrally charged.
How do stain-free gels compare to other protein dyes?
Protein visualization data obtained from stain-free gels are comparable to those obtained from gels stained with other dyes. In general, the sensitivity of stain-free gels is equal to that of Coomassie-stained gels for all proteins.
What is the range of protein load in a stain-free gel?
Stain-free gels provide a linear dynamic range between 10 and 80 µg of total protein load from cell or tissue lysates at a higher range of protein load (Figure 3A), and from 20 to 1 µg at a lower range (Figure 3B) ( Taylor et al. 2013, Hammond et al. 2013 ). Fig 3.
How do mini-protean TGX stain-free gels work?
The trihalo compounds in Mini-PROTEAN TGX Stain-Free Gels react with tryptophan residues in proteins in a UV-induced 1-minute reaction to produce fluorescence.
Can stain-free gels be used with chemiluminescence and fluorescent western blotting?
Stain-Free gels are fully compatible with both chemiluminescence and fluorescent western blotting detection methods. For chemiluminescence detection, acquire blot image on Stain-Free enabled imager just before applying ECL reagent to achieve the most accurate representation of total protein present.