Expert Coffee Chat Webinar: Western Blotting and Protein Electrophoresis

HTML
Consult with an Specialist

Have a Western Blotting Question?

Ask a Specialist

Our customers asked our Bio-Rad application scientists about the current issues most relevant to their protein electrophoresis and western blotting projects. A lively discussion was had about imaging, antibodies, fluorescent western blotting, housekeeping proteins, and more!

Air Date: May 20, 2020

We had so many great questions asked by the audience. Watch the whole video, or quickly jump to a specific question:

  • 00:10  Introduction
  • 3:10  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 β-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 kDa for a while. I optimized it using shorter transfer time and smaller pores of filter and yet got smear 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

Western Blotting Questions Answered by Our Experts

In this section, you will find audience questions and thorough answers from our application scientists grouped by common topics.

Protein Sample Preparation

 What is your advice on obtaining a quantitative western blot for proteins that we believe are aggregating?

 Is western blot a good way to determine integrin (heterodimer integrins) concentration? Specifically, does western ?blotting cause integrins to separate into their subunits (and should you detect those subunits instead of the heterodimer) or do heterodimer integrins generally stay intact for antibody detection

 I have issues with protein concentration. I use Bio-Rad protein assay reagent with a spectrophotometer. With the readings I get, I prepare protein samples. But my GAPDH is always off. This is faced by most of our labmates. Could it be a problem with spectrophotometer calibration? Which is the best method for protein concentration detection?

Protein Electrophoresis

 How do you prevent smiley faces on gels?

 Do you have any experience in using tris-tricine gels and transblot turbo / small peptides?

 Western blotting is a real toxic technique requiring several toxic reagents, such as acrylamide. Is it possible to perform western blotting without using toxic reagents, such as acrylamide?

 Do you have tips to avoid smileys in blots and also to avoid merging of bands?

 Can I run my gels at 180 V for 1 hour? My gel is 4–15% precast gel. My protein of interest is between 35–70 kDa.

 If I have equal protein concentrations in each lane but a different volume, could this cause problems with running?

 In tissue lysate westerns, sometimes individual wells will run very wide or very narrow on the same gel. Any idea what could cause this?

Western Blotting Transfer

 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?

 I've been struggling with WB of small molecular protein, like 16–22 kDa for a while. I optimized it using shorter transfer time and smaller pores of filter and yet got smear band. Can you give me some advice?

 If I am experiencing excessive protein left on my gels when transferring to a nitrocellulose membrane, how would I go about trouble shooting this issue to improve the transferring process?

 How do you determine the best blocking solution to use for your western blot (nonfat milk, BSA, casein, etc)?

 Tips on choosing the best buffer type for transfer of proteins?

 Which membranes are best for which application - PVDF and nitrocellulose?

 What is the best method to remove Ponceau S red?

 Is the image of the gel before and after blotting and image of blot a good sustitute for Ponceau S?

 In a western transfer, sometimes the lower MW proteins transfer completely, but the larger proteins are still in the gel. How can I ensure that all of the proteins are transferred completely?

 Is there a difference between PVDF and nitrocellulose membranes? How do you choose the best membrane for your assay?

 I've been struggling with the transfer to a PVDF membrane from a 1.0 mm polyacrylamide gel in a Trans-Blot Turbo because the proteins wouldn"t transfer completely efficient to the membrane; any advice?

 While doing transfer, how much ever I try to get rid of bubble, sometimes bubbles screw the experiment. Is there any trick?

 I have been using semi-dry transfer and facing bubble issue. Is there any specific amount of transfer buffer that I should use to avoid bubbles?

Immunodetection

 How many times can a primary antibody be used?

 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!

 What is the best way to avoid losing your signal after stripping and reporting?

 Any advice on reducing/eliminating doublets? Are blots with doublets generally disregarded as accurate?

 Any tips on using western blot to detect highly glycosylated proteins?

 Our lab can never fully remove actin once we run it, no matter how long we strip the membrane. It will always show up back on our film.

 Would it be advisable to use multiple primary antibodies for protein detection ? If yes, then how would that affect sensitivity and specifity?

 If my target protein is rabbit primary, after developing the blot, can I probe the blot with mouse primary antibody without stripping the blot?

 What would be the ideal conditions for stripping a really strong target?

 I have been trying to detect a small protein (9 KDa), using the pure protein I have no problem detecting it, but I can"t detect it from the cell lysate. Any advice?

 Besides GAPDH, b-actin, and a-tubulin, are there other housekeeping genes you recommend? I've observed that a lot of the primary antibodies for housekeeping genes are very close to the size of the target protein.

 How important and what is the best concentration of Tween 20 in the wash buffer and in the antibodies solution?

 I've been trying to detect a protein that has been known to be hard to detect. Do you have any advice or troubleshooting tips?

 Should I have a separate secondary for each primary or is it OK to use the same secondary for multiple primaries?

 While using femto and pico substrate, can I use pico first and if my signal is not good, can I again use femto substrate without washing?

 I am probing my membranes for UCP1, which appears being very strong on my membrane. This results in the stripping buffer incompletely removing the antibody to reprobe for beta-actin. As they are very close in MW, it"s hard to pre-cut the membrane to avoid this. What would you suggest?

Western Blot Imaging

 Is film more sensitive and a better way to perform western blot imaging?

 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?

 Do you have tips for how to decrease the blots" background?

 How can I avoid saturated/less exposure signals with different expression levels?

 Do you have advice on how to pick the right housekeeping protein for your experiment?

 How do we know that the interaction in stain free technology doesn"t affect protein integrity downstream like binding of antibody?

 Which molecular weight marker do you recommend for fluoresence multiplexing?

 Since there are so many different methods out there, which method is best when quantifying proteins from a western blot?

 Any advice on getting rid of ghost bands on film?

 Any tricks for getting a clear stain free blot image? I have found that mine never look like the examples, they are always very faint.

 If you use stain free, can you then cut the membranes?

 How are stain-free gels visualized? I have an infrared imager and a fluorescent imager. Can either of these detect the total protein on a stain-free gel?

 I used to cut my gel and blot from different antibodies, but now seems that is not correct?

Miscellaneous Western Blotting

 Is western blotting a quantitative technique? What is the general method to do quantitative western blotting?

 Why are western blots so challenging?

 Can you explain single cell western blotting?

 Do you guys have any experience for (IP and/or WB) glycoproteins? In my case, sialylation.

 Can I use different concentrations of the same sample on same gel as replicates for relative quantification?

Coffee Chat with Bio-Rad Application Scientists

Bi-Weekly Webinar Series – Every other Wednesday at 9:00 AM PDT (17:00 BST)

Join our Application Scientists for an informal chat about the most relevant issues for
our customers, answers to Tech Support questions, new products, tips for success
in your work, and live Q&A.

View All Coffee Chat Topics

Related Products

Western Blotting Products

  • Stain-Free Western Workflow

    Stain-Free Western Workflow

    Our Stain-Free Western Workflow System allows you to quickly check electrophoresis and blot transfer quality and obtain truly quantitative western blotting results, updating traditional blotting techniques with innovative tools.

  • Imaging Systems and Software

    Imaging Systems & Software

    ChemiDoc Imagers offer best-in-class performance with ease of use for fluorescence and chemiluminescence detection and all general gel documentation applications.

  • Western Blotting Trans-Blot Turbo Transfer System

    Western Blotting Trans-Blot Turbo Transfer System

    The Trans-Blot Turbo System enables fast, efficient, and reproducible transfer of proteins up to 400 kD. Preassembled Trans-Blot Turbo Transfer Packs are optimized for superior blot transfer.

  • Western Blotting Bluffers & Reagents

    Browse our full line of western blotting reagents:

    • Antibodies and Conjugates
    • Chemiluminescence Detection Reagents
    • Buffers and Blocking Agents
  • View All Western Blotting Products »

Electrophoresis Products

Western Blotting Resources

FEATURED WEBINARUnderstand the Factors that are Crucial to Successful Western Blotting

Successful Western Blotting

Join Bio-Rad western blotting expert Kenneth Oh and his guest speakers for a webinar series that explores the many factors that go into the design and execution of successful and repeatable western blots. Chemiluminescence or fluorescence, qualitative or quantitative western blot, not only will we discuss the how-tos, we'll take a deeper dive and discuss the whys.

View Webinar

 
  • Stain-Free Western Blot Imaging

    See the fast, convenient, and transparent V3 Western Workflow process, how Image Lab Software performs data normalization using total protein loading controls, and how stain-free imaging yields reliable western blot results.

  • Can We Trust Western Blots?

    In this presentation, Aldrin Gomes, Associate Professor of Neurobiology at UC Davis, talks about key success factors to instill trust in western blotting data.

  • Single-Cell Western Blot and Stain-Free Total Protein Loading Control

    Demonstration of quantitative analysis of fiber type–specific protein expression patterns in single muscle cells with a unique western blotting protocol utilizing Bio-Rad's stain-free imaging technology.

 
 
  • Western Blotting Techniques

    Western Blotting Techniques

    Learn more about western blotting techniques. Find step-by-step protocols and helpful tips on equipment, membranes, transfer conditions, and detection methods.

  • Protein Separation and Analysis

    Protein Separation and Analysis

    Bio-Rad's V3 Western Workflow facilitates speed and validation at each step of a western blotting experiment — from running gels to quantifying proteins.

  • Protein Electrophoresis

    Protein Electrophoresis

    Find protocols, video tutorials, and selection guides to help you at every step of your electrophoresis experiments.

  • Imaging and Analysis

    Imaging and Analysis

    Find information on protein visualization and quantitation methods, gel and blot imaging instrumentation, and image analysis software.

 

Better Western Blotting Guide

Tips, Techniques, and Technologies from the Western Blotting
Experts at Bio‑Rad Laboratories