TidyBlot, a Western blot detection reagent

TidyBlot

Western blot detection reagent

Upcoming IP webinar: "Complex or not?"

Presented by: Dr Rachael Preston (Development Scientist at Bio-Rad Laboratories)

Tidy up your IP/Western blot with one simple reagent change


TidyBlot (STAR209P) is Bio-Rad’s brand new HRP conjugated Western blot detection reagent and offers key benefits over standard heavy and light chain secondary antibodies.


TidyBlot performance

TidyBlot Western blot detection reagent:HRP specifically binds to native (non-reduced) antibodies. In contrast to conventional secondary antibodies, TidyBlot therefore enables the detection of immunoblotted target protein bands, without the interference from denatured IgG (Figure 1, lane 2).

Sources of denatured IgG include endogenous IgGs present in cell/tissue lysates (e.g. from spleen and liver) and primary antibodies released from beads during immunoprecipitation experiments. The latter are particularly obstructive when Western blot detection of (co-)immunoprecipitated proteins is desired as IgG heavy (~50 kDa) and light chains (~25 kDa) may mask the protein(s) of interest.


TidyBlot advantages

TidyBlot’s advantages compared to conventional heavy and light chain secondary antibodies are:

  • Detects only what matters - TidyBlot exclusively binds to native non-denatured antibodies and not to any IgGs present in your immunoprecipitate or lysate. Avoid obstruction by IgG heavy and light chains (Figure 1, lane 5) and produce background free Western blots (Figure 1, lane 2).
  • Is convenient – just substitute your conventional secondary antibody for TidyBlot.
  • Offers broad species coverage – TidyBlot detects a variety of monoclonal and polyclonal antibodies (Figure 2). No need to buy several HRP conjugated secondary antibodies.

Fig 1.Comparison of HRP conjugated TidyBlot Western blot detection reagent (lanes 2 & 3) to a standard anti-mouse IgG (heavy & light chain) secondary antibody (lanes 5 & 6). In contrast to the anti-mouse secondary antibody, TidyBlot only binds to the mouse anti-human actin gamma antibody (VMA00049) used for detection (lane 2). Lanes 3 and 6 are no primary antibody controls.
Please note that TidyBlot is HRP conjugated and that the Western blot bands (red) and protein standards (green) have been pseudocolored.

Fig 2Detection of goat, rabbit and sheep IgG polyclonal antibodies with TidyBlot. HeLa (YAP1 and mTOR) and Hek293 (NFkappaB p65) cell lysates were run on SDS PAGE and transferred onto PVDF membranes. Goat anti-YAP1 (VPA00104), rabbit anti-mTOR (VPA00174) and sheep anti-NFkappaB p65 (VPA00015) antibodies were added at a dilution of 1/1000. Detection was performed with TidyBlot at a 1/200 dilution. All primary antibodies are part of the Western blot validated PrecisionAb™ range. 
Please note that TidyBlot is HRP conjugated and that the Western blot bands (red) and protein standards (green) have been pseudocolored.


Overview of TidyBlot compatibility

TidyBlot detects the IgG monoclonal and polyclonal antibodies listed below:

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

If using mouse IgG1 monoclonal antibodies, perform a dot blot to determine compatibility. TidyBlot Western blot detection reagent:HRP 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. We also offer a comparable product for rabbit; mouse anti-rabbit light chain, clone SB62a (MCA6003P) is an HRP conjugated secondary antibody that does not bind to rabbit IgG heavy chains.


TidyBlot solves two major problems:
 

TidyBlot problem

Problem 1

When performing Western blot detection of IP samples:

  • Protein of interest/immunoprecipitated protein has a molecular weight of ~50 kDa  -  heavy-chain will obstruct detection of protein of interest
  • Protein of interest/immunoprecipitated protein  has a molecular weight of ~25 kDa - light-chain will obstruct detection of protein of interest
TidyBlot solution

Solution - TidyBlot

As TidyBlot only binds to native non-denatured antibodies immunoprecipiated proteins of 50 kDa and 25 kDa can be easily visualized without any obstruction.


TidyBlot problem

Problem 2

When performing Western blot detection of tissue samples rich in endogenous immunoglobulins:

  • Additional bands from binding of secondary antibody to endogenous immunoglobulins
  • High background from unspecific secondary antibody binding
TidyBlot solution

Solution - TidyBlot

As TidyBlot only binds to native non-denatured antibodies trouble-free Western blot detection of tissue lysates can be performed (low background risk)

 

Summary - use TidyBlot for the generation of clean publication quality Western blot data