Key Product Details

Validated by

Knockout/Knockdown, Biological Validation

Species Reactivity

Validated:

Human, Mouse, Rat, Amphibian, Primate

Cited:

Human, Mouse, Rat

Applications

Validated:

Knockout Validated, Immunohistochemistry, Immunohistochemistry-Paraffin, Immunohistochemistry-Frozen, Western Blot, Flow Cytometry, Flow (Intracellular), Immunocytochemistry/ Immunofluorescence, Chromatin Immunoprecipitation (ChIP)

Cited:

Immunohistochemistry, Immunohistochemistry-Paraffin, Immunohistochemistry-Frozen, Western Blot, Immunocytochemistry/ Immunofluorescence, Chemotaxis, IF/IHC, Knockdown Validated

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all BMAL1 Primary Antibodies »

Product Specifications

Immunogen

Bacterially expressed human BMAL1 (amino acids 392-626). [UniProt# O00327].

Reactivity Notes

Mouse reactivity reported in scientific literature (PMID:32732906). Amphibian reactivity reported from a verified customer review.

Localization

Nuclear

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

70 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Description

Novus Biologicals Knockout (KO) Validated Rabbit BMAL1 Antibody - BSA Free (NB100-2288) is a polyclonal antibody validated for use in IHC, WB, Flow, ICC/IF and ChIP. Anti-BMAL1 Antibody: Cited in 75 publications. All Novus Biologicals antibodies are covered by our 100% guarantee.

Scientific Data Images for BMAL1 Antibody - BSA Free

Western Blot: BMAL1 Antibody [NB100-2288]

Western Blot: BMAL1 Antibody [NB100-2288]

BMAL1-Antibody-Western-Blot-NB100-2288-img0021.jpg
Immunohistochemistry: BMAL1 Antibody [NB100-2288]

Immunohistochemistry: BMAL1 Antibody [NB100-2288]

Immunohistochemistry: BMAL1 Antibody [NB100-2288] - Analysis of BMAL1 in mouse cerebral cortex (left: wt, right: Bmal1 KO) using anti-BMAL1 antibody. Image from verified customer review.
Immunohistochemistry: BMAL1 Antibody [NB100-2288]

Immunohistochemistry: BMAL1 Antibody [NB100-2288]

BMAL1-Antibody-Immunocytochemistry-Immunofluorescence-NB100-2288-img0024.jpg
Western Blot: BMAL1 Antibody [NB100-2288]

Western Blot: BMAL1 Antibody [NB100-2288]

BMAL1-Antibody-Western-Blot-NB100-2288-img0023.jpg
Immunocytochemistry/ Immunofluorescence: BMAL1 Antibody [NB100-2288]

Immunocytochemistry/ Immunofluorescence: BMAL1 Antibody [NB100-2288]

BMAL1-Antibody-Immunocytochemistry-Immunofluorescence-NB100-2288-img0022.jpg
Flow (Intracellular): BMAL1 Antibody [NB100-2288]

Flow (Intracellular): BMAL1 Antibody [NB100-2288]

Flow (Intracellular): BMAL1 Antibody [NB100-2288] - An intracellular stain was performed on HeLa cells with NB100-2288AF647(blue) and a matched isotype control (orange). Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 5 ug/mL for 30 minutes at room temperature. Both antibodies were conjugated to Alexa Fluor 647.
Knockout Validated: BMAL1 Antibody [NB100-2288]

Immunohistochemistry: BMAL1 Antibody [NB100-2288]

BMAL1-Antibody-Knockout-Validated-NB100-2288-img0020.jpg
Flow Cytometry: BMAL1 Antibody [NB100-2288]

Flow Cytometry: BMAL1 Antibody [NB100-2288]

Flow Cytometry: BMAL1 Antibody [NB100-2288] - An intracellular stain was performed on Jurkat cells with NB100-2288 and a matched isotype control. Cells were fixed with 4% PFA and then permeabilized with 0.1% saponin. Cells were incubated in an antibody dilution of 2.5 ug/mL for 30 minutes at room temperature, followed by Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody.
Western Blot: BMAL1 Antibody [NB100-2288]

Western Blot: BMAL1 Antibody [NB100-2288]

Western Blot: BMAL1 Antibody [NB100-2288] - Analysis of BMAL1 in A) MCF7, B) NIH/3T3, C) PC12.
Western Blot: BMAL1 Antibody [NB100-2288]

Western Blot: BMAL1 Antibody [NB100-2288]

Western Blot: BMAL1 Antibody [NB100-2288] - Analysis of MOP3 on 3T3/L1 cell lysates.
Immunocytochemistry/ Immunofluorescence: BMAL1 Antibody [NB100-2288]

Immunocytochemistry/ Immunofluorescence: BMAL1 Antibody [NB100-2288]

Immunocytochemistry/Immunofluorescence: BMAL1 Antibody [NB100-2288] - BMAL1 antibody was tested in HeLa cells at a 1:200 dilution against Dylight 488 (Green). Alpha tubulin and nuclei were counterstained against DyLight 568 (Red) and DAPI (Blue), respectively.
Immunohistochemistry: BMAL1 Antibody [NB100-2288]

Immunohistochemistry: BMAL1 Antibody [NB100-2288]

Immunohistochemistry: BMAL1 Antibody [NB100-2288] - Analysis of BMAL1 in mouse brain using DAB with hematoxylin counterstain.
Immunohistochemistry-Paraffin: BMAL1 Antibody [NB100-2288]

Immunohistochemistry-Paraffin: BMAL1 Antibody [NB100-2288]

Immunohistochemistry-Paraffin: BMAL1 Antibody [NB100-2288] - Analysis of a FFPE tissue section of mouse brain using 1:200 dilution of rabbit anti-BMAL1 antibody. The staining was developed using HRP labeled anti-rabbit IgG secondary antibody and DAB reagent, and nuclei of cells were counter-stained with hematoxylin. This BMAL1 antibody generated a specific nuclear staining in most of the cells and a relatively weaker cytoplasmic signal was also observed.
BMAL1 Antibody

Immunohistochemistry-Frozen: Rabbit Polyclonal BMAL1 Antibody [NB100-2288] -

Immunohistochemistry-Frozen: Rabbit Polyclonal BMAL1 Antibody [NB100-2288] - IHC-fixed frozen in the brain of green tree frogs. DAB staining, 1:1000 dilution. Image from a verified customer review.
BMAL1 Antibody

BMAL1 in MCF7 Human Cell Line.

BMAL1 was detected in immersion fixed MCF7 human breast cancer cell line using Rabbit anti-BMAL1 Affinity Purified Polyclonal Antibody conjugated to Alexa Fluor® 647 (Catalog # NB100-2288AF647) (light blue) at 10 µg/mL overnight at 4C. Cells were stained counterstained with DAPI (blue). Cells were imaged using a 40X objective.
BMAL1 Antibody

Immunocytochemistry/ Immunofluorescence: BMAL1 Antibody [NB100-2288] -

Immunocytochemistry/ Immunofluorescence: BMAL1 Antibody [NB100-2288] - Rapamycin reduces the accumulation of BMAL1 in Per2 knockout miceA. As shown in the left panel, tissue samples from Per2 knockout mice (mPER−/−) depict robust accumulation of nuclear BMAL1 (arrow) compared to control littermates (arrowhead)(*** p<0.001). Administration of Rapamycin reduces the accumulation of BMAL1 in the epidermis of mPer−/− mice (arrowhead) compared to mPer−/− mice receiving vehicle alone (** p<0.01) to levels comparable to wild-type mice receiving vehicle alone (ns: p>0.05). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27285754), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
BMAL1 Antibody

Western Blot: BMAL1 Antibody [NB100-2288] -

Western Blot: BMAL1 Antibody [NB100-2288] - Small interference RNA targeting Raptor & Rictor disrupts BMAL1 accumulation in HNSCCTargeted disruption of Raptor (A-B) & Rictor (C-D) using siRNA results in a dose-dependent downregulation of BMAL1 in HNSCC cells. E. Disruption of PTEN by protein oxidation causes activation of mTOR signaling, resulting in accumulation of BMAL1. Notably, inhibition of mTOR signaling, particularly mTORC1 & mTORC2, results in restoration of normal BMAL1 levels in the epidermis of mice & head & neck cancer cells. These results demonstrate a novel role for mTOR in regulating nuclear levels of the core clock gene BMAL1. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27285754), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
BMAL1 Antibody

Western Blot: BMAL1 Antibody [NB100-2288] -

Western Blot: BMAL1 Antibody [NB100-2288] - Targeted disruption of PTEN in vitro & in vivo induces activation of pS6 & BMAL1A. & B. Targeted inhibition of PTEN using siRNA results in concentration-dependent inhibition of PTEN protein in HNSCC cells. HNSCC cells show accumulation of BMAL1 & pS6 in response to PTEN inhibition. C. Immunofluorescence assay to detect BMAL1 in PTEN conditional knockout mice (K14cre PtenF/F) & control littermates. Immunofluorescences & graphic show high accumulation of nuclear BMAL1 in K14cre PtenF/F mice compared to control mice (K14cre). Scale bars represent 50 μm. Black & white images depict cells positive for BMAL1 in K14cre PtenF/F mice (arrows) compared to few BMAL-positive cells in control mice (arrowhead). Scale bars represent 10 μm. (**p<0.01). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27285754), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
BMAL1 Antibody

Western Blot: BMAL1 Antibody [NB100-2288] -

Western Blot: BMAL1 Antibody [NB100-2288] - Small interference RNA targeting Raptor & Rictor disrupts BMAL1 accumulation in HNSCCTargeted disruption of Raptor (A-B) & Rictor (C-D) using siRNA results in a dose-dependent downregulation of BMAL1 in HNSCC cells. E. Disruption of PTEN by protein oxidation causes activation of mTOR signaling, resulting in accumulation of BMAL1. Notably, inhibition of mTOR signaling, particularly mTORC1 & mTORC2, results in restoration of normal BMAL1 levels in the epidermis of mice & head & neck cancer cells. These results demonstrate a novel role for mTOR in regulating nuclear levels of the core clock gene BMAL1. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27285754), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
BMAL1 Antibody

Western Blot: BMAL1 Antibody [NB100-2288] -

Western Blot: BMAL1 Antibody [NB100-2288] - Small interference RNA targeting Raptor & Rictor disrupts BMAL1 accumulation in HNSCCTargeted disruption of Raptor (A-B) & Rictor (C-D) using siRNA results in a dose-dependent downregulation of BMAL1 in HNSCC cells. E. Disruption of PTEN by protein oxidation causes activation of mTOR signaling, resulting in accumulation of BMAL1. Notably, inhibition of mTOR signaling, particularly mTORC1 & mTORC2, results in restoration of normal BMAL1 levels in the epidermis of mice & head & neck cancer cells. These results demonstrate a novel role for mTOR in regulating nuclear levels of the core clock gene BMAL1. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27285754), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
BMAL1 Antibody

Western Blot: BMAL1 Antibody [NB100-2288] -

Western Blot: BMAL1 Antibody [NB100-2288] - Small interference RNA targeting Raptor & Rictor disrupts BMAL1 accumulation in HNSCCTargeted disruption of Raptor (A-B) & Rictor (C-D) using siRNA results in a dose-dependent downregulation of BMAL1 in HNSCC cells. E. Disruption of PTEN by protein oxidation causes activation of mTOR signaling, resulting in accumulation of BMAL1. Notably, inhibition of mTOR signaling, particularly mTORC1 & mTORC2, results in restoration of normal BMAL1 levels in the epidermis of mice & head & neck cancer cells. These results demonstrate a novel role for mTOR in regulating nuclear levels of the core clock gene BMAL1. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27285754), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
BMAL1 Antibody

Western Blot: BMAL1 Antibody [NB100-2288] -

Western Blot: BMAL1 Antibody [NB100-2288] - Targeted disruption of PTEN in vitro & in vivo induces activation of pS6 & BMAL1A. & B. Targeted inhibition of PTEN using siRNA results in concentration-dependent inhibition of PTEN protein in HNSCC cells. HNSCC cells show accumulation of BMAL1 & pS6 in response to PTEN inhibition. C. Immunofluorescence assay to detect BMAL1 in PTEN conditional knockout mice (K14cre PtenF/F) & control littermates. Immunofluorescences & graphic show high accumulation of nuclear BMAL1 in K14cre PtenF/F mice compared to control mice (K14cre). Scale bars represent 50 μm. Black & white images depict cells positive for BMAL1 in K14cre PtenF/F mice (arrows) compared to few BMAL-positive cells in control mice (arrowhead). Scale bars represent 10 μm. (**p<0.01). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27285754), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
BMAL1 Antibody

Western Blot: BMAL1 Antibody [NB100-2288] -

Western Blot: BMAL1 Antibody [NB100-2288] - Oxidation causes accumulation of BMAL1A. Head & neck cancer cells have different expression levels of the core clock protein BMAL1. B. Immunofluorescence assay depicts accumulation of ROS (green channel) & BMAL1 (red channel) upon oxidation. C. & D. Western blot assay demonstrates time-dependent accumulation of BMAL1 upon oxidative stress in HNSCC cells (*p<0.05, **p<0.01). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27285754), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
BMAL1 Antibody

Immunocytochemistry/ Immunofluorescence: BMAL1 Antibody [NB100-2288] -

Immunocytochemistry/ Immunofluorescence: BMAL1 Antibody [NB100-2288] - Targeted disruption of PTEN in vitro & in vivo induces activation of pS6 & BMAL1A. & B. Targeted inhibition of PTEN using siRNA results in concentration-dependent inhibition of PTEN protein in HNSCC cells. HNSCC cells show accumulation of BMAL1 & pS6 in response to PTEN inhibition. C. Immunofluorescence assay to detect BMAL1 in PTEN conditional knockout mice (K14cre PtenF/F) & control littermates. Immunofluorescences & graphic show high accumulation of nuclear BMAL1 in K14cre PtenF/F mice compared to control mice (K14cre). Scale bars represent 50 μm. Black & white images depict cells positive for BMAL1 in K14cre PtenF/F mice (arrows) compared to few BMAL-positive cells in control mice (arrowhead). Scale bars represent 10 μm. (**p<0.01). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/27285754), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
BMAL1 Antibody

Immunohistochemistry: BMAL1 Antibody [NB100-2288] -

Immunohistochemistry: BMAL1 Antibody [NB100-2288] - Astrocyte Bmal1 regulates genes with conflicting effects on A beta deposition. (A) Topro, GFAP, & BMAL1 staining in CA1 hippocampus of 4-month-old BMAL1 aKO; APP/PS1-21 mice & Cre- controls (scale bar = 100 µm). Arrows indicate astrocyte nuclei quantified as indicated by Topro nuclei surrounded by GFAP positivity. Blue circles indicate nuclei quantified as BMAL1 negative. Quantification of astrocytes counted as BMAL1- or BMAL1+ is shown on the right. n = 5 mice per group, **** = p < 0.0001 by two-way ANOVA with Sidak multiple comparisons test. (B) Heatmap of Fluidigm qPCR analysis of 20 genes involved in the circadian clock, glial activation, & Alzheimer’s Disease in cortex from Aldh1l1-CreERT2; Bmal1fl/fl mice & Cre- controls with or without APP/PS1-21 or APPNL-G-F/wt (n = 6–8 mice per group). Two-way ANOVA analysis: c = significant main effect of Cre genotype, m = main effect of A beta model, c*m = interaction effect of cre & A beta model, - = no significance (all p < 0.05). (C) Individually plotted genes from A. * = p < 0.05, ** = p < 0.005, *** = p < 0.0005 by two-way ANOVA with Sidak multiple comparisons test. Panel B was made using GraphPad Prism version 9.2 (https://www.graphpad.com). Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/35110643), licensed under a CC-BY license. Not internally tested by Novus Biologicals.

Applications for BMAL1 Antibody - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:100

Immunohistochemistry

1:250. Use reported in scientific literature (PMID 33510438)

Immunohistochemistry-Frozen

reported in scientific literature (PMID 23736292)

Immunohistochemistry-Paraffin

1:250

Western Blot

0.5 ug/mL - 2 ug/mL
Application Notes
In ICC/IF, primarily nuclear staining was observed with weak cytoplasmic staining in MCF7 cells. In Western Blot, a band was observed ~70 kDa. In IHC-P, staining was observed in the nuclei of mouse brain tissue. Prior to immunostaining paraffin tissues, antigen retrieval with sodium citrate buffer (pH 6.0) is recommended. The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Reviewed Applications

Read 2 reviews rated 4 using NB100-2288 in the following applications:

Flow Cytometry Panel Builder

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Advanced Features

  • Spectra Viewer - Custom analysis of spectra from multiple fluorochromes
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  • Antigen Density Selector - Match fluorochrome brightness with antigen density
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Flow Cytometry

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C for up to 3 months. For longer storage, aliquot and store at -20C.

Background: BMAL1

BMAL1 (brain and muscle ARNT-like 1; ARNTL) is an essential core component in circadian clock machinery which is regulatory mechanism for circadian rhythms. Circadian clock genes include three period proteins (PER1, PER2 and PER3), two cryptochromes (CRY1 and CRY2), CLOCK, NPAS2 and BMAL proteins, wherein BMAL1 has a potential to heterodimerize with CLOCK or NPAS2 genes; and this neocomplex drives transcription from E-box elements (5'-CACGTG-3') found in circadian-responsive genes's promoters. PER/CRY proteins negatively regulate CLOCK/BMAL1 dimer-mediated transcription, thereby forming the feedback loop that regulates the timing of clock gene transcription. BMAL1 also associates with GNB2L1/RACK1 and PRKCA in a nuclear complex, whertein GNB2L1 and PRKCA are recruited to the complex in a circadian manner. BMAL1 undergoes acetylation (Lys-538), phosphorylation and sumoylation (Lys-259) upon dimerization with CLOCK and acetylation facilitates CRY1-mediated repression. CLOCK-BMAL1 double mutations within PAS domains leads to syngernistic desensitization to high levels of CRY on repression of CLOCK-BMAL1 transcriptional activity of PER1 and, disrupt circadian rhythmicity. Clock genes functions primarily as tumor suppressors and their abbarant expression is observed in malignant pleural mesothelioma, colorectal and breast cancer.

Long Name

Brain and Muscle ARNT-Like 1

Alternate Names

ARNTL, BHLHE5, BMAL1c, JAP3, MOP3, PASD3, TIC

Gene Symbol

BMAL1

Additional BMAL1 Products

Product Documents for BMAL1 Antibody - BSA Free

Certificate of Analysis

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Product Specific Notices for BMAL1 Antibody - BSA Free

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Citations for BMAL1 Antibody - BSA Free

Customer Reviews for BMAL1 Antibody - BSA Free (2)

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Customer Images

Showing  1 - 2 of 2 reviews Showing All
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  • BMAL1 Antibody
    Name: Deborah Lutterschmidt
    Application: Immunohistochemistry-Frozen
    Sample Tested: Brain
    Species: Hyla cinerea
    Verified Customer | Posted 10/20/2023
    IHC- fixed frozen in the brain of green tree frogs. DAB staining, 1:1000 dilution
    BMAL1 Antibody - BSA Free NB100-2288
    Bio-Techne Response
    This review was submitted through the legacy Novus Innovators Program, reflecting a new species or application tested on a primary antibody.
  • Name: Anonymous
    Application: Immunofluorescence
    Sample Tested: mouse brain
    Species: Mouse
    Verified Customer | Posted 06/29/2015
    Bmal1 staining in mouse cerebral cortex: wt and Bmal1 KO
    BMAL1 Antibody - BSA Free NB100-2288

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Protocols

View specific protocols for BMAL1 Antibody - BSA Free (NB100-2288):

Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.

Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes (keep slides in the sodium citrate buffer at all times).

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in PBS for 5 minutes.
3. Block each section with 100-400 ul blocking solution (1% BSA in PBS) for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4 C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul HRP polymer conjugated secondary antibody. Incubate 30 minutes at room temperature.
7. Wash sections three times in wash buffer for 5 minutes each.
8. Add 100-400 ul DAB substrate to each section and monitor staining closely.
9. As soon as the sections develop, immerse slides in deionized water.
10. Counterstain sections in hematoxylin.
11. Wash sections in deionized water two times for 5 minutes each.
12. Dehydrate sections.
13. Mount coverslips.

Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturer's instructions.

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