Western Blotting Flashcards
What is the impact of low-grade/impure methanol in transfer buffer?
Impure methanol can increase transfer buffer conductivity and yield a poor transfer.
Transfer Buffer Formulations…BioRad
Can methanol be substituted in transfer buffer?
In many cases, yes (with ethanol). There is minimal impact with transfer, but check with your samples.
Transfer Buffer Formulations…BioRad
Can you resuse transfer buffer?
No. The buffer will likely lose its ability to maintain a stable pH during transfer (emperically though, yes).
Transfer Buffer Formulations…BioRad
Can you dilute transfer buffers below recommended levels?
No. It decreases their buffering capacity.
Transfer Buffer Formulations…BioRad
Is pH important for transfer buffers?
Yes. Do not adjust the pH unless specifically indicated. Adjusting the pH can result in increased buffer conductivity, manifested by higher intial current output and decreased resistance.
Transfer Buffer Formulations…BioRad
What high SDS (above recipe) do to transfer buffer?
Increasing SDS increases protein transferfrom the gel, but decreases binding of the protein to nitocellulose membrane. PVDF should be used instead when SDS is is used in the transfer buffer.
Transfer Buffer Formulations…BioRad
What does addition of SDS do to transfer buffer?
Adding SDS increases relative current, power and heating during transfer. It may also affect antigenicity of some proteins.
Transfer Buffer Formulations…BioRad
What does increasing methanol percentage do to transfer buffer?
Increasing MeOH decreases protein transfer from the gel and increases binding of the protein to the nitrocellulose membrane.
Bis-Tris vs Tris-Glycine buffers
They have different charge shielding properties – aka they have different migration property of charged species within an electric field. Best to use like with like (can use MOPS- or MES-based running buffer with bis-tris).
Bis-Tris has longer shelf life.
BCA
- Bichinchoninic Acid Assay
- colorimetric reaction to quantify proteins in solution, specifically: Cu+2 reacts with protein –> Cu+ (purple)
- reduction of Cu+2 occurs with cysteine, cystine, tryptophan, tyrosine and peptide bonds
- reaction is temperature dependent
- best with complex protein mixtures (ex. total cell lysates)
https://www.researchgate.net/post/Nanodrop-or-BCA-Bradford-assay
Methods in Enyzmology, 2021, chapter 17
Nanodrop for protein quantification
- measures the absorbance generated by aromatic aminoacids in your proteins (highly dependent on the percentage of these aminoacids in your sample and how well your standard reflects this percentage)
- aromatic aminoacids: tryptophan, tyrosine, phenylalanine
- best for aminoacid composition in purified protein samples
Problems with transfer? Consider the buffer and…
…and preincubate the gel in transfer buffer (5min)
…remember PVDF must first be activated in 30sec in blotting methanol and rinses with DI water beforehand
