Peptide Blocking Protocol

Peptide blocking
protocol for
immunostaining(IHC, ICC, IF)
By Alomone Labs

What are blocking peptides?

When are they used?

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 immunohistochemistry (IHC),
immunocytochemistry (ICC) and immunofluorescent
staining (IF) experiments. Note that they are not suitablefor use as a control for applications requiring live andintact cells (flow cytometry, live cell imaging).

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

Schematic:
Immunostaining Peptide
Blocking Protocol

Slide 1

In a control sample, pre-incubation of a primary antibody
with an excess of blocking peptide will occupy all theantibody binding sites. This prevents subsequent binding
to the epitope in the protein of interest you are looking
to detect. A comparison of this staining (antibody +blocking peptide) versus a sample when you use theantibody alone, can help you determine non-specificbinding. The staining that disappears when you use theantibody + blocking peptide, is specific to that antibody.

Slide 2++AntibodyAlone

Antibody+ Blocking peptide

Slide 1

Slide 2

Final result following secondary
antibody and detection

Peptide Blocking Protocol

Materials and Reagents• Antibody dilution buffer: Usually PBST (Phosphatebuffered saline + 0.1% Tween 20) with 1% BSA• Blocking (immunizing) peptide• Two tubes• Two identical test samples (Two identical slides
containing the tissue or cells of interest)• Antibody

Method
1. Complete the slide preparation, fixation andpermeabilization (for intracellular antigens) of yourtissue or cells as per your immunostaining protocol.
Refer to our IHC protocol or ICC/IF protocols forfurther information. Proceed to the stage below,
where your primary antibody is prepared, with orwithout pre-absorption with blocking peptide.
2. Reconstitute your lyophilized blocking peptide insterile PBS or double distilled water (DDW) as perinstructions (for example at 150 µg/ml). Ensure
there is complete dissolution and triturate asnecessary.
3. Optimize your IHC or ICC/IF protocol in advance.
Follow the product guidelines, or previous
experimental optimizations for a recommended
dilution for your primary antibody. Determine thequantity of antibody required for two experiments(e.g If your optimal antibody concentration is at
2 µg/ml, you will require 2 µg of antibody if you
are using 1 ml buffer, for each of your slides).
4. Dilute the necessary amount of antibody in dilution
buffer to the final volume needed for the two
experiments (4 µg in 2 ml in this example).
5. Divide this equally into two tubes.
6. Label the first tube “+peptide”. Add ten times excessof blocking peptide to antibody (e.g If your peptide
was at a concentration of 150 µg/ml, you would
need to add 133 µl to give 20 µg total peptide in
1 ml buffer in this example).

7. In the second tube, labeled “antibody alone”, add anequivalent amount of buffer volume (133 µl in thisexample).
8. Incubate both tubes, with agitation overnight at 4°C,
or alternatively at room temperature for 30 min.
9. Perform your IHC or ICC/IF antibody staining
protocol on the two identical sample slides, using
the antibody + blocking peptide (labeled “+peptide”) for one slide as your primary antibody
source, and the antibody alone for the other. 		
Ensure you know which is which by labeling 		the slides.
10. Proceed with PBS washes as per your protocol toremove unbound primary, and apply thesecondary antibody for detection, repeat washingprocedure.
11. Mount your slides as per your protocol.
12. Observe and compare the staining patternobtained in the two test slides. The staining 		that disappears when using the blocked antibody
is specific to the antibody. Other staining that isvisible represents non-specific binding.

Peptide Blocking Protocol

IHC/ICC/IF: Published customer data using
Alomone Labs blocking peptide
Figure 1: ICC using Anti-TRPV4 Antibody (#ACC-034)
with blocking peptide
Immunocytochemical staining of mouse mCCDcl1
kidney cells using Anti-TRPV4 Antibody (#ACC-034),(green). TRPV4 staining is completely abolished when
the antibody is preincubated with the TRPV4 BlockingPeptide (#BLP-CC034) (+ BP in right panel).

TRPV4

TRPV4 + BP+ DAPI+ DAPI

Adapted from Li, Y. et al. (2016) PLoS ONE 11,e0155006 with permission of PLoS.

Figure 2: IHC using Anti-CaV2.3 AntibodyA(#ACC-006) with blocking peptide

Immunohistochemical staining of mouse accessory
olfactory bulb sections using Anti-CaV2.3 (CACNA1E) Antibody (#ACC-006). A(i). CaV2.3 staining(green) is detected in mitral cells. A(ii). Highmagnification of (i). A(iii). Note the absence ofimmunofluorescent staining when the antibody ispreincubated with CaV2.3/CACNA1E Blocking(i)

Peptide (#BLP-CC006).

Adapted from Gorin, M. et al. (2016) J. Neurosci. 36,
3127 with permission of the Society for Neuroscience.
(iii)(ii)