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TidyBlot

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TidyBlot Western Blot Detection Reagent thumbnail image 1 TidyBlot Western Blot Detection Reagent thumbnail image 2 TidyBlot Western Blot Detection Reagent thumbnail image 3 TidyBlot Western Blot Detection Reagent thumbnail image 4
TidyBlot Western Blot Detection Reagent gallery image 1
25 μg of HeLa cell lysate spiked with 3.5 μg of mouse IgG was run in lanes 2, 3, 5 and 6 under reducing conditions on SDS PAGE using the Bio-Rad V3 system and transferred to PVDF membrane. Bio-Rad Precision Plus MW marker was run in lanes 1 and 4. Mouse anti actin gamma (VMA00049) was used to detect actin gamma (approximate MW of 46 kDa) in lanes 2 and 5 only. Lanes 3 and 6 were used as controls to detect the background signaling of the secondary detection agent (no primary antibody control). TidyBlot Western Blot Detection Reagent:HRP was used as the secondary reagent in lanes 2 and 3 and an HRP conjugated anti-mouse IgG heavy and light chain antibody was used as the secondary in lanes 5 and 6. The Blot was visualized with the Bio-Rad Chemidoc™ Touch Imaging System. The TidyBlot Western Blot Detection Reagent:HRP only bound to the native actin gamma antibody whereas the standard anti-Mouse IgG heavy and light recognized both the native and denatured antibodies
TidyBlot Western Blot Detection Reagent gallery image 2
HeLa (lane 2) cell lysate was run under reducing conditions on SDS PAGE using the Bio-Rad V3 system and transferred to a PVDF membrane. Bio-Rad Precision Plus molecular weight marker was run in lane 1. Rabbit anti Human GAPDH (AHP1628) was used as the primary antibody at a dilution of 1/1000 in lane 2. TidyBlot was used at 1/200 as the secondary detection reagent. Visualization was carried out using the Bio-Rad ChemiDoc with automatic exposure.
TidyBlot Western Blot Detection Reagent gallery image 3
Western blot analysis of GAPDH IP samples. IP was performed on Jurkat cell lysates using 10 μg (lanes 1 & 7), 5 μg (lanes 2 & 8), 1 μg (lanes 3 & 9) Mouse anti GAPDH antibody (MCA4739), and 10 μg Mouse IgG1 Negative Control (lanes 4 & 10) (MCA1209).

8.75 μl of each IP was loaded onto an AnykD™ Criterion™ TGX Stain-Free™ gel. 8.75 µl Jurkat whole cell lysate (WCL) was run in lanes 5 & 11 while Precision Plus Protein™ Prestained Standards were run in lane 6.

Rabbit anti GAPDH antibody (VPA00187) was used at 1/1000 in lanes 1-5. HRP conjugated Tidyblot (STAR209P) was used at 1/200 and visualized on the Bio-Rad Chemidoc™ Touch Imaging System. Arrow points to GAPDH (molecular weight 37 kDa).
TidyBlot Western Blot Detection Reagent gallery image 4
Mouse thymus lysate (25 μg) was loaded onto a 4–15% Criterion™ TGX Stain-Free™ Protein Gel (lanes 2, 3, 5, and 6) and proteins were transferred onto a nitrocellulose membrane. Bio-Rad Precision Plus Protein™ Prestained Standards were run in lanes 1 and 4. Mouse anti actin gamma (MCA5776GA) was used to detect actin gamma (approximate MW of 46 kDa) in lanes 2 and 5 only. Lanes 3 and 6 were used as controls to detect the background signaling of the secondary detection reagent (no primary antibody control). HRP-conjugated goat anti mouse IgG (heavy and light chain) secondary antibody (STAR207P) was used at 1/10,000 in lanes 2 and 3. TidyBlot Western Blot Detection Reagent:HRP (STAR209P) was used as the secondary reagent at 1/200 in lanes 5 and 6. The blot was visualized with the ChemiDoc™ MP Imaging System.

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TidyBlot Western Blot Detection Reagent:HRP

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Accessory Reagent
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TidyBlot

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Technical Suppport
Product Code Applications Pack Size List Price Your Price Qty
STAR209P
Datasheet Datasheet Datasheet
SDS Safety Datasheet SDS
WB * 0.5 ml
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STAR209PA
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SDS Safety Datasheet SDS
WB * 1 ml
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STAR209PT
Datasheet Datasheet Datasheet
SDS Safety Datasheet SDS
WB * 50 µl
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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 interference from denatured IgG. Sources of denatured IgG include endogenous IgGs present in cell/tissue lysate and primary antibodies released from beads during immunoprecipitation experiments. The latter are particularly obstructive when detecting of (co-)immunoprecipitated proteins as IgG heavy (~50 kDa) and light chains (~25 kDa) may mask the protein(s) of interest.

N.B. Reactivity of TidyBlot Western Blot Detection Reagent:HRP to mouse IgG1 can be variable. Bio-Rad recommend customers assess compatibility with their IgG1 antibodies via dot blot assay.

Preservative Stabilisers
<0.06% ProClin™ 300
Regulatory
For research purposes only
Guarantee
12 months from date of despatch
Acknowledgements
Proclin™ 300 is a trademark of The Dow Chemical Company (“Dow”) or an affiliated company of Dow.

Store at +4oC. DO NOT FREEZE.
This product should be stored undiluted. Should this product contain a precipitate we recommend microcentrifugation before use.

This product has been reported to work in the following applications. This information is derived from testing within our laboratories, peer-reviewed publications or personal communications from the originators. Please refer to references indicated for further information. For general protocol recommendations, please visit the antibody protocols page.
Application Name Verified Min Dilution Max Dilution
Western Blotting 1 1/40 1/400
  1. 1 TidyBlot Western Blot Detection Reagent:HRP 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 denaturing the immunoprecipitate/lysate fully (consider the duration of boiling the sample to reduce IgG disulphide bonds and the type/concentration of reducing agent).
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures.
Technical Advice
Note: If using mouse IgG1, perform a dot blot to determine compatibilty. TidyBlot Western Blot Detection Reagent:HRP might not detect mouse IgG1.

TidyBlot Western Blot Detection Reagent:HRP detects the IgG polyclonal and monoclonal 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
Instructions For Use
When using a 1/200 dilution (25 μl in 5 ml milk protein), TidyBlot Western Blot Detection Reagent:HRP is sufficient to perform approximately 20 (STAR209P) or 40 (STAR209PA) mini Western Blots.

1. Dilute primary antibody (see technical advice section) to appropriate concentration in blocking buffer
2. Incubate blot with primary antibody
3. Wash blot
4. Dilute TidyBlot Western Blot Detection Reagent:HRP For best results, optimize dilutions for each experiment
5. Incubate blot with TidyBlot Western Blot Detection Reagent:HRP
6. Wash blot
7. Prepare and add HRP substrate according to manufacturers instructions
8. Develop blot
When using a 1/200 dilution (25 μl in 5 ml milk protein), TidyBlot Western Blot Detection Reagent:HRP is sufficient to perform approximately 20 (STAR209P) or 40 (STAR209PA) mini Western Blots.

1. Dilute primary antibody (see technical advice section) to appropriate concentration in blocking buffer
2. Incubate blot with primary antibody
3. Wash blot
4. Dilute TidyBlot Western Blot Detection Reagent:HRP For best results, optimize dilutions for each experiment
5. Incubate blot with TidyBlot Western Blot Detection Reagent:HRP
6. Wash blot
7. Prepare and add HRP substrate according to manufacturers instructions
8. Develop blot
When using a 1/200 dilution (25 μl in 5 ml milk protein), TidyBlot Western Blot Detection Reagent:HRP is sufficient to perform approximately 2 (STAR209PT), 20 (STAR209P) or 40 (STAR209PA) mini Western Blots.

1. Dilute primary antibody (see technical advice section) to appropriate concentration in blocking buffer
2. Incubate blot with primary antibody
3. Wash blot
4. Dilute TidyBlot Western Blot Detection Reagent:HRP For best results, optimize dilutions for each experiment
5. Incubate blot with TidyBlot Western Blot Detection Reagent:HRP
6. Wash blot
7. Prepare and add HRP substrate according to manufacturers instructions
8. Develop blot

Description Product Code Applications Pack Size List Price Your Price Quantity
Rat anti Mouse Kappa Light Chain:HRP MCA152P E WB 0.1 mg loader
List Price Your Price
loader
Description Rat anti Mouse Kappa Light Chain:HRP
Mouse anti Rabbit Light Chain:HRP MCA6003P E WB 0.5 ml loader
List Price Your Price
loader
Description Mouse anti Rabbit Light Chain:HRP

References for TidyBlot

  1. Inoue, S.et al. (2019) Drebrin-like (Dbnl) Controls Neuronal Migration via Regulating N-Cadherin Expression in the Developing Cerebral Cortex.
    J Neurosci. 39 (4): 678-91.
  2. Kråkenes, T. et al. (2019) CDR2L Is the Major Yo Antibody Target in Paraneoplastic Cerebellar Degeneration.
    Ann Neurol. 86 (2): 316-21.
  3. Wu, J. et al. (2019) Androgen receptor SUMOylation regulates bone mass in male mice.
    Mol Cell Endocrinol. 479: 117-122.
  4. Welkenhuysen, N.et al. (2018) Robustness of Nutrient Signaling Is Maintained by Interconnectivity Between Signal Transduction Pathways.
    Front Physiol. 9: 1964.
  5. Zuliani, I. et al. (2020) The Dysregulation of OGT/OGA Cycle Mediates Tau and APP Neuropathology in Down Syndrome.
    Neurotherapeutics. Nov 30 [Epub ahead of print].
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