Protein Block (ab64226)

Submit a review Q&A (4)References (8)

Overview

  • Product name

    Protein Block

  • Tested applications

    Suitable for: IHC-P, ELISA, WBmore details

  • General notes

    Protein blocking buffer ab64226 for serum-free blocking of non-specific antibody binding in IHC, ELISA and western blot. Ready to use. No mixing or dilution required.

    For use in ELISA, add Protein Block to well and incubate for 2-10 minutes before addition of sample. Wash and continue procedure. For use in western blotting, incubate for one hour at room temperature or overnight at 4°C.

     

    IHC protocol suitable for use with Protein Block ab64226:
    For frozen sections, skip steps 1 and 2. For fluorescent IHC, skip step 3, incubate with fluorescent dye conjugated secondary at step 6, skip rest of steps, and mount with anti-fade mounting medium.

    1. Deparaffinize and rehydrate formalin-fixed paraffin-embedded tissue section.

    2. Use appropriate antigen retrieval buffer or enzyme (primary antibody dependent) to treat sections. Wash 3 times in buffer.

    3. Add enough hydrogen peroxide blocking solution to cover the sections. Incubate for 10 minutes. Wash 2 times in buffer. If necessary, use avidin biotin blocking.

    4. Apply Protein Block ab64226 (or normal serum from same species as secondary antibody) and incubate for 5 minutes at room temperature to block nonspecific background staining. Wash once in buffer.

    5. Apply primary antibody in antibody diluent and incubate. Wash 4 times in buffer.

    6. Incubate with biotinylated secondary antibody (or HRP polymer secondary antibody and skip step 7). Wash 4 times in buffer. 

    7. Apply streptavidin-HRP and incubate for 10 minutes at room temperature.

    8. Rinse 4 times in buffer. Place slide in DAB substrate or AEC substrate and incubate until desired color is achieved. Rinse 4 times in buffer.

    9. Add enough drops of hematoxylin to cover the section. Incubate for 1 minute.

    10. Rinse 7-8 times in tap water. Add mounting medium to cover the section.


    Find complete IHC kits, and reagents for antigen retrieval, blocking, signal amplification, visualization, counterstaining, and mounting in the IHC kits and reagents guide.

Properties

  • Form

    Liquid

  • Storage instructions

    Store at +4°C.

  • Storage buffer

    pH: 7.40
    Preservative: 0.1% Sodium azide
    Constituents: PBS, 0.5% Casein, 0.5% BSA
  • Concentration information loading...

  • Reagent notes

    ab64226 reduces the handling of animal serums in the laboratory. The need to match species with the secondary antibody is eliminated due to the lack of normal serum in this product. It has been shown to be effective for immunohistochemistry, ELISA, and blot and requires no mixing or diluting.

  • Research areas

  • Relevance

    Protein Block is used with immunolabeling techniques for the reduction of non-specific background staining.

Applications

Our Abpromise guarantee covers the use of ab64226 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P Use at an assay dependent dilution.
ELISA Use at an assay dependent dilution.
WB Use at an assay dependent dilution.

Images

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Protein Block (ab64226)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Protein Block (ab64226)

    ab64226 was used as pasr of the ab93677 anti-Mouse and Rabbit specific HRP (ABC) Detection IHC kit to perform immunohistochemical analysis of Human Tonsil tissue labeling Ki-67.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Protein Block (ab64226)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Protein Block (ab64226)Yu S.H., PLoS One 9 (12), Fig 7c. doi: 10.1371/journal.pone.0114649 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    ab64226 was used in the mouse and rabbit specific HRP/DAB detection IHC Kit (ab64264) to visualize Immunohistochemical analysis staining CD163 in mouse soleus muscle section. Samples were incubated in rabbit CD163 antibody at 1:450 dilution.

References

This product has been referenced in:

  • Nishimori S  et al. PTHrP targets HDAC4 and HDAC5 to repress chondrocyte hypertrophy. JCI Insight 4:N/A (2019). Read more (PubMed: 30843886) »
  • Salomão NG  et al. BALB/c mice infected with DENV-2 strain 66985 by the intravenous route display injury in the central nervous system. Sci Rep 8:9754 (2018). Read more (PubMed: 29950590) »
See all 8 Publications for this product

Publishing research using ab64226? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-4 of 4 Abreviews or Q&A

Question

is ab64226 the same protein block that is in ab80437?

Read More
Answer

ab64226 is the protein block used in ab80437. A link to ab64226 is below:

Read More

Question

ab128988:
What is the compositions of the protein block? Does it contain BSA?
Can the block be used in ICC?

Read More
Answer

Thank you for contacting us.
The lab sent me the following information:
1. What is the compositions of the protein block? Does it contain BSA?
ANSWER: PBS solution, pH 7.6, with 0.5% BSA, 0.5% casein and less than 0.1% sodium azide
2. Can the block be used in ICC?
ANSWER: The product has not been tested in immunocytochemistry applications.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Question

I need to develop double IHC analysis on formalin-fixed paraffin-embedded (FFPE) tissue sections of human tumors specimens, and after we have to do laser capture microdissection of positive cells.   I already have two primary antibodies directed against CD44 (produced in mouse) and against ALDH1 (produced in rabbit).   I'm looking for double staining kit (or all of the individual components which analysis needs) with alcohol insoluble chromogens because after IHC and before laser cature microdissection I have to dehydrate the slides.   Thank you for your time and consideration.

Read More
Answer

Thank you for contacting us.

We do not currently offer double staining IHC kits. Your staining conditions are challenging as, of the two common HRP chromogens, only DAB is alcohol insoluable. While of the common AP chromagens, only BCIP/NBT is insoluable. This means that to do double labeling you will have to do a sequential stain first using one detection system and then with the other.

I have collected a list, with links, of the reagents which you would need for this type of staining.

1) After de-parrafinizing and re-hydrating the samples, perform an antigen retrieval step treating with Cirate buffer pH6.0,https://www.abcam.com/10x-Citrate-Buffer-pH-6-0-ab64214.html, in the microwave or pressure cooker. After performing the antigen retrieval rinse in cold tap water for 10 minutes.

2) Rinse slides in TBST,https://www.abcam.com/20x-TBS-T-with-Tween-20-ab64250.html.

3) Performing a blocking step using protein block,https://www.abcam.com/Protein-Block-ab64226.html.

4) Incubate sections with your first primary antibody, for clarity I will assume the CD44 antibody, raised in mouse.

5) Wash with TBST.

6) Block endogenous peroxidases using aHydrogen Peroxide Blocking Reagent,https://www.abcam.com/Hydrogen-Peroxide-Blocking-Reagent-ab94666.html.

7) Incubate your tissue with a biotinylated secondary antibody, in this instance anti-Mouse IgG,https://www.abcam.com/Biotinylated-Goat-anti-Mouse-IgG-H-L-Ready-to-Use-ab64255.html.

8) Wash with TBST.

9) Detect your first antibody usingStreptavidin Peroxidase (Ready to Use)https://www.abcam.com/Streptavidin-Peroxidase-Ready-to-Use-ab64269.html. Note that this does not containa serum or BSA solution as diluent. Serum may contain biotin therefore competing Streptavidin binding with biotinylated secondary antibody, therefore reducing binding activity.

10) Apply your chromogen, DABsubtrate,https://www.abcam.com/DAB-Substrate-Kit-ab64238.html.

11) Wash with TBST.

12)Performing a second blocking step using protein block,https://www.abcam.com/Protein-Block-ab64226.html.

13)Wash with TBST.

14)Incubate sections with the your second primary antibody, this time ALDH1, raised in rabbit.

15)Wash with TBST.

16)Incubate your tissue with a biotinylated secondary antibody, in this instance anti-Rabbit IgG,https://www.abcam.com/Biotinylated-Goat-Anti-Rabbit-IgG-H-L-Ready-to-use-ab64256.html

17)Wash with TBST.

18)Detect your first antibody usingStreptavidin Alkaline Phosphatase (Ready to Use)https://www.abcam.com/Streptavidin-Alkaline-Phosphatase-Ready-to-Use-ab64268.html.

19) Apply your second chromogenAlkaline Phosphatase chromogen (BCIP/NBT) - Ready to Use,https://www.abcam.com/Alkaline-Phosphatase-chromogen-BCIP-NBT-Ready-to-Use-ab7468.html.

18) Wash with TBST, counterstain if desired.


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Question

Last week I asked Janice White, who is responsible for the purchasing, to order an Abcam antiboty (CD31, ab28364) and protein block (ab64226). But the products were delivered last Friday when she was not in her office  and she gave me an email on the arrival of the products on Monday. Unfortunately, I was out of the own on Monday so I got those on Tuesday. These products should be shipped and kept in 4C. But those are stored in room temperature when I got them.   Your data sheet on antibody says majority of antibodies are stable at room temperature for at least one week. Can CD30 antibody apply to this situation? And how about the protein block?   As you know, the quality of antibodies are very very important. Please give me an answer.  

Read More
Answer

Most of our antibodies will remain stable for 7 days at room temperature. For ab28364, anti-CD31, it should still be viable and work as expected. As for the protein block, we have no information on how long it will remain viable at room temperature, but I think it would still work for you, although I cannot guarantee that it will still work since it was not stored correctly. In future when you place orders, you can specify a delivery date so that you can avoid a delivery arriving when there will not be someone available to receive and store the products as required. If there is anything else I can do, please let me know.

Read More

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com