Peptide Blocking Protocol

Peptide blocking
protocol forWesternblotting (WB)
By Alomone Labs

What are blocking peptides?
A comparison of this staining (antibody + blockingpeptide) versus a sample when you use the antibodyalone, can help you determine non-specific binding.
The staining that disappears when you use theantibody + blocking peptide, is specific to that antibody.

When are they used?

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SDS - PAGELaThe inclusion of a blocking peptide as an experimental
control is recommended, to help validate antibody
specificity.

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WB Peptide Blocking Protocol
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Blocking peptides comprise the original peptide antigen
used in immunization to raise a primary antibody.
Consequently, they are sometimes referred to as
immunizing peptides. We have also previously referredto them at Alomone Labs as ‘negative control antigens’.
They correspond to the epitope (specific amino-acidsequence recognized by an antibody) on a target
protein. Blocking peptides can be used to compete or
block antibody binding, and are therefore a valuable
control for antibody specificity in Western blotting (WB)
and immunostaining experiments.

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In a control sample, pre-incubation of an antibody withan excess of blocking peptide will occupy all theantibody binding sites. This prevents subsequentbinding to the epitope in the protein of interest you arelooking to detect.

++Antibody
Alone

Antibody+ Blocking
peptide

95
72
55
3628

Final result following
secondary antibodyand detection

Peptide Blocking Protocol

Materials and Reagents• Blocking buffer: Usually TBST (Tris-buffered saline,0.1% Tween® 20) with either 5% non-fat dry milk,
or 1% BSA + 0.05% NaN3
• Antibody blocking (immunizing) peptide• Two tubes• Two identical test samples. (Two lanes in WB withtransfer membrane cut into strips to separate thesamples for incubation)• AntibodyMethod
1. Reconstitute your lyophilized blocking peptide insterile PBS or double distilled water (DDW) as
per instructions (for example at 150 µg/ml).
Ensure there is complete dissolution and triturate
as necessary.
2. Optimize your WB protocol. Refer to our WB
protocol for more information. Follow the productguidelines, or previous experimental optimizations
for a recommended dilution for your primary
antibody. Determine the quantity of antibody
required for two experiments.
3. If your optimal antibody concentration is a 1:200
dilution, add 20 µg antibody to one 1.5 ml
eppendorf tube, and 500 µl 1% BSA in PBS. Labelit “antibody alone”

7. Perform your WB staining protocol as normal on
the two identical samples, using the antibody +blocking peptide (labeled "+ peptide") for one
test strip as your primary antibody source, andthe antibody alone for the other. Ensure you
know which is which.
8. Proceed with thorough washes as per your WB
protocol to remove unbound primary, and apply
your secondary antibody for detection, repeat
washing procedure.
9. Develop your blots, observe and compare thestaining obtained in the two test strips. The bandthat disappears when using the blocked antibody
is specific to the antibody. Other visible bands
represent non-specific binding.

To a second identical tube, add 20 µg antibody,
20 µg negative control antigen (supplied with theantibody) and 500 µl 1% BSA in PBS. Label it“+peptide”.
4. Incubate both tubes for 1 hr at room temperature.
5. Transfer the content of each eppendorf to larger
tubes and add 4.5 ml 1% BSA in PBS with 0.1%
Tween® -20 and 0.05% NaN3 to each. Add thecontent of each tube to its respective test strip
membrane for parallel experiments.
6. Incubate both tubes, with gentle rocking,overnight at 4°C.

(NB: If multiple bands disappear, in addition to the one at the anticipated MW of your protein, it is important to consider the following:
If the MW is higher than expected - could your protein of interest have post-translational modifications?
If the MW is lower than expected - could your protein of interest have a truncated form, or could it have degraded in your sample?)

Peptide Blocking Protocol

WB: Published customer data using Alomone Labs
blocking peptide controls

CF-7
MMSCIN

Western blot analysis of human plasmacytoma(INA-6) cells, human mesenchymal stem cell (MSC),
and human breast cancer cell lysates using Anti-KISS1
Receptor (extracellular) Antibody (#AKR-001), (upperpanels). The band observed is completely eradicatedwhen the antibody is preincubated with KISS1
Receptor (extracellular) Blocking Peptide (#BLPKR001).

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Figure 1: WB using Anti-KISS1 Receptor(extracellular) Antibody (#AKR-001) with blocking
peptideBlocking (-)
Blocking (+)

Right panels show the band remains when antibodies
were preincubated with the unrelated P2X4
Receptor Blocking Peptide (#BLP-PR004) (+pep4r).
Adapted from Jimenez-Pacheco, A.et al. (2016)J.
Neurosci. 36, 5920 with permission of the Society
for Neuroscience.

+- pep7r + pep7r

P2X7R
Actin
Human- pep7r + pep7r

pep4r

Western blots show a P2X7R band (left panels) inepileptic mouse and human hippocampal samples (n= 1/lane) and elimination of the band (middle panels)
when Anti-P2X7 Receptor Antibody (# APR-004)
was preincubated with P2X7 Receptor Blocking
Peptide (#BLP-PR004) (+pep7r).

Mouse+Figure 2: WB using Anti-P2X7 Receptor Antibody(# APR-004) with blocking peptide

pep4r

Adapted from Dotterweich, J. et al. (2016) PLoS
ONE 11, e0155087 with permission of PLoS.

P2X7R
Actin