TidyBlot, a Western blot detection reagent

TidyBlot

Frequently Asked Questions

We have collected a set of frequently asked questions about TidyBlot Western blot detection reagent:

TidyBlot Western Blot detection reagent: Frequently Asked Questions

TidyBlot Western Blot detection reagent: Frequently Asked Questions

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  1. What is TidyBlot Western blot detection reagent:HRP?
  2. What are TidyBlot’s benefits over a conventional secondary antibody?
  3. What is the recommended dilution range?
  4. How do I use TidyBlot and what should I be aware of?
  5. What antibody types is TidyBlot compatible with?
  6. What if I want to use TidyBlot with a mouse IgG1 antibody?
  7. How many blots can I do with one vial of TidyBlot?
  8. Can I purchase TidyBlot conjugated to other enzymes or fluorophores?
  9. Can I purchase TidyBlot in a bigger size or in bulk?
  10. Why are the TidyBlot Western blot data in color when the reagent is HRP conjugated?
  11. What other related secondary detection reagents does Bio-Rad offer?
  12. Where can I find information about Bio-Rad’s IP and Western blot tested antibodies?
     
1

What is TidyBlot Western blot detection reagent:HRP?

TidyBlot (STAR209P) is a proprietary HRP conjugated secondary detection reagent, which only detects native IgG antibodies. TidyBlot is not an antibody but can be used like a secondary antibody in Western blot experiments.

2

What are TidyBlot’s benefits over a conventional secondary antibody?

TidyBlot has three advantages compared to conventional HRP conjugated (heavy and light chain) secondary antibodies.

  • Detects only what matters – TidyBlot exclusively binds to native non-denatured antibodies and not to any IgGs present in your immunoprecipitate or lysate. You can therefore detect your protein of interest without obstruction by IgG heavy and light chains, and produce background free Western blots.
  • Convenience – just substitute your conventional secondary antibody for TidyBlot.
  • Broad species compatibility – TidyBlot detects a variety of monoclonal and polyclonal antibodies. No need to buy several HRP conjugated secondary antibodies.
3

What is the recommended dilution range?

TidyBlot is a substitute for an HRP conjugated secondary antibody and is therefore used in the same manner. The recommended TidyBlot dilution range is 1/40 to 1/400 (depending on the abundance of the protein of interest and the substrate used). In our experience a 1/200 dilution of TidyBlot in 5% non-fat milk yields more than 20 mini Western blots when using Bio-Rad’s Clarity™ Western ECL blotting substrate.

4

How do I use TidyBlot and what should I be aware of?

TidyBlot preferentially binds to the native antibody used during the Western blot procedure rather than the SDS-denatured/reduced form present in lysates/immunoprecititates. Best results are therefore achieved by fully denaturing the immunoprecipitate/lysate (consider the duration of sample boiling to reduce IgG disulphide bonds and the type/concentration of reducing agent). TidyBlot is used in the same manner as conventional HRP conjugated secondary antibodies.

5

What antibody types is TidyBlot compatible with?

TidyBlot is compatible with the below listed IgG monoclonal and polyclonal antibodies:

Species

Monoclonal Isotype(s)

Polyclonal

Bovine IgG2 Compatible
Goat IgG2 Compatible
Human IgG1, IgG2, IgG4 Compatible
Mouse IgG2a, IgG2b, IgG3
IgG1: affinity for IgG1 varies and may not be strong

Affinity should therefore be tested on an antibody by antibody basis by performing dot blot analysis.
Compatible
Rat IgG2c Compatible
Rabbit Total IgG Compatible
Sheep IgG2 Compatible

TidyBlot is not known to detect IgA, IgD, IgE and IgM antibodies.

6

What if I want to use TidyBlot with a mouse IgG1 antibody?

If using a mouse IgG1 antibody, perform dot blot analysis to determine compatibility. The TidyBlot might not detect mouse IgG1. In that case you could try our HRP conjugated rat anti-mouse kappa light chain specific antibody, clone OX-20 (MCA152P). This antibody does not detect mouse IgG heavy chains and thereby allows you to detect immunoprecipitated proteins of ~50 kDa without obstruction by heavy chains.

7

How many blots can I perform with one vial of TidyBlot?

One vial of TidyBlot (0.5ml) enables you to perform at least 20 mini Western blots; assuming a 1/200 dilution in 5% non-fat milk using Bio-Rad’s Clarity™ Western ECL blotting substrate. As is necessary for every secondary detection reagent, you will need to establish the optimal dilution by performing a dilution series.

8

Can I purchase TidyBlot conjugated to other enzymes or fluorophores?

TidyBlot is currently only available conjugated to HRP. Unfortunately, we do not offer TidyBlot in other formats.

9

Can I purchase TidyBlot in a bigger size or in bulk?

Yes, TidyBlot can be purchased in bulk as a multiple of 0.5 ml. Please contact your local sales representative for a bulk quote and delivery time.

10

Why are the TidyBlot Western blot data in color when the reagent is HRP conjugated?

TidyBlot Western blot data have been generated using the Bio-Rad ChemiDoc™ MP System rather than X-ray film. The system has various filters for the detection of fluorophores and other detection reagents based on their excitation and emission wavelengths. Each reagent is assigned a color (red, green or blue) when visualizing the blot with the ChemiDoc imaging software. In the case of the TidyBlot data, the chemi channel was used to detect HRP chemiluminescence and assigned the color red. Protein standards (Bio-Rad Precision Plus Protein™ All Blue Protein Standards) were detected in the Dylight® 650 channel and assigned the color green*.

*Please note that these are pseudocolors and are not a reflection of emission colors.

11

What other related secondary detection reagents does Bio-Rad offer?

Bio-Rad offers an HRP conjugated rat anti-mouse kappa light chain specific antibody, clone OX-20 (MCA152P). This antibody does not detect mouse IgG heavy chains and thereby allows you to detect immunoprecipitated proteins of ~50 kDa without obstruction by heavy chains. We also offer a comparable product for rabbit; mouse anti-rabbit light chain specific antibody, clone SB62a (MCA6003P), which is an HRP conjugated secondary antibody that does not bind to rabbit IgG heavy chains.

12

Where can I find information about Bio-Rad’s IP and Western blot tested antibodies?

For more information, please go to www.bio-rad-antibodies.com/tidyblot, contact your local sales representative or our technical support team.


 


DyLight® is a trademark of Thermo Fisher Scientific Inc. and its subsidiaries.
ChemiDoc™ MP System, Clarity™ Western ECL blotting substrate, Precision Plus Protein™ All Blue Protein Standards and PrecisionAb™ are trademarks of Bio-Rad Laboratories, Inc.