Join us for a chat with Bio-Rad application scientists about the current issues most relevant to your research. Jump to specific topics by following the links below.
In this episode, we discuss: Western Blot Optimization
- Get rid of your western blot ghosts by reducing background
- Keep your gel from saying "cheese" and smiling
- Make that band shine as bright as a star by increasing your signal to noise
We had so many great audience questions. Watch the whole video or quickly jump to a specific question:
00:10 Introduction
2:47 Is western blotting a quantitative technique? What is the general method to do quantitative western blotting?
6:17 Is film more sensitive and a better way to perform western blotting imaging?
10:50 How many times can a primary-antibody be used?
13:25 How crucial is it to do the Ponceau S Stain? And what is the best strategy for removing the stain prior to adding the blocking solution? And how many times can you strip the membrane to use for different protein detection?
18:23 When imaging different proteins one as experimental and one as a loading control (such as iNOS and beta-Actin), which have been separated from the same membrane by cutting, should they be imaged at the same time or separately? Or should they even be probed for on two separate blots?
22:40 Do you have tips for how to decrease the blots' background?
29:15 How do you prevent smiley faces on gels?
33:20 I am a new lab tech working with fluorescent western blots for the first time. The antibody we are using is clearly labeling a singular protein that is not our target protein. Could this be a problem with the secondary we are pairing with the primary? Could it be a problem with the buffer we are using with the primary? Any troubleshooting advice is appreciated!
39:09 Good morning, I've been struggling with WB of small molecular protein, like 16–22 ka for a while. I optimized it using shorter transfer time and smaller pores of filter and yet got a smeared band. Can you give me some advice?
41:23 If I am experiencing excessive protein left on my gels when transferring to a nitrocellulose membrane, how would I go about troubleshooting this issue to improve the transferring process?
44:04 How to avoid saturated/less exposure signals with different expression levels
48:34 Do you have advice on how to pick the right housekeeping protein for your experiment?
50:18 Why are western blots so challenging?
56:30 Closing
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