Overview

  • Product name
  • Description
    Rabbit polyclonal to Rad51
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IP, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Chicken, Human, Xenopus laevis
  • Immunogen

    Purified recombinant full length protein (Human)

  • Positive control
    • HeLa cell extract. Normal human diploid cells.
  • General notes

    Please click here for Instructions For Use/Protocol.

Properties

Applications

Our Abpromise guarantee covers the use of ab63801 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
WB 1/2000 - 1/10000. Detects a band of approximately 37 kDa (predicted molecular weight: 37 kDa).
IP Use at an assay dependent dilution.
ICC/IF 1/100.

Target

  • Function
    Plays an important role in homologous strand exchange, a key step in DNA repair through homologous recombination. Binds to single and double-stranded DNA and exhibits DNA-dependent ATPase activity. Catalyzes the recognition of homology and strand exchange between homologous DNA partners to form a joint molecule between a processed DNA break and the repair template. Binds to single-stranded DNA in an ATP-dependent manner to form nucleoprotein filaments which are essential for the homology search and strand exchange (PubMed:26681308). Part of a PALB2-scaffolded HR complex containing BRCA2 and RAD51C and which is thought to play a role in DNA repair by HR. Plays a role in regulating mitochondrial DNA copy number under conditions of oxidative stress in the presence of RAD51C and XRCC3.
  • Tissue specificity
    Highly expressed in testis and thymus, followed by small intestine, placenta, colon, pancreas and ovary. Weakly expressed in breast.
  • Involvement in disease
    Breast cancer
    Mirror movements 2
    Defects in RAD51 are found in a patient with microcephaly, mental retardation without bone marrow failure and pediatric cancers.
  • Sequence similarities
    Belongs to the RecA family. RAD51 subfamily.
    Contains 1 HhH domain.
  • Domain
    The nuclear localization may reside in the C-terminus (between 259 and 339 AA).
  • Post-translational
    modifications
    Ubiquitinated by the SCF(FBXO18) E3 ubiquitin ligase complex, regulating RAD51 subcellular location and preventing its association with DNA.
    Phosphorylated. Phosphorylation of Thr-309 by CHEK1 may enhance association with chromatin at sites of DNA damage and promote DNA repair by homologous recombination. Phosphorylation by ABL1 inhibits function.
  • Cellular localization
    Nucleus. Cytoplasm. Cytoplasm, perinuclear region. Mitochondrion matrix. Cytoplasm, cytoskeleton, microtubule organizing center, centrosome. Colocalizes with RAD51AP1 and RPA2 to multiple nuclear foci upon induction of DNA damage. DNA damage induces an increase in nuclear levels. Together with FIGNL1, redistributed in discrete nuclear DNA damage-induced foci after ionizing radiation (IR) or camptothecin (CPT) treatment. Accumulated at sites of DNA damage in a SPIDR-dependent manner.
  • Information by UniProt
  • Database links
  • Alternative names
    • BRCA1/BRCA2 containing complex, subunit 5 antibody
    • BRCC 5 antibody
    • BRCC5 antibody
    • DNA repair protein RAD51 homolog 1 antibody
    • DNA repair protein rhp51 antibody
    • FANCR antibody
    • hRAD51 antibody
    • HsRAD51 antibody
    • HsT16930 antibody
    • MRMV2 antibody
    • Rad 51 antibody
    • RAD51 antibody
    • RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae) antibody
    • RAD51 homolog A antibody
    • RAD51 homolog antibody
    • RAD51 recombinase antibody
    • RAD51, S. cerevisiae, homolog of antibody
    • RAD51_HUMAN antibody
    • RAD51A antibody
    • RECA antibody
    • RecA like protein antibody
    • RecA, E. coli, homolog of antibody
    • Recombination protein A antibody
    see all

Images

  • 2BN hTert (XLF-deficient) human fibroblasts were fixed for 30 min with methanol at −20°C, dipped for 1 min in ice cold acetone for permeabilization and washed three times for 10 min with PBS/1% FCS. Non-specific antigens were blocked for 30 min in 5% BSA in PBS/1% FCS. Samples were incubated with ab63801 (1∶15000) in PBS/1% FCS over night at 4°C, washed three times in PBS/1% FCS and incubated for 1 h at room temperature with AlexaFluor 488 conjugated secondary antibodies (1 : 500). After three times of washing in PBS, cells were DAPI stained.

    Image shows 2BN hTert (XLF-deficient) human fibroblasts analyzed 2 h post IR with 1 Gy.

  • Anti-Rad51 antibody (ab63801) at 1/1000 dilution + Crude extract of Xenopus

    Predicted band size: 37 kDa

  • Immunocytochemistry/ Immunofluorescence analysis of human osteosarcoma U2OS cells X-ray irradiation before and 2h after, immunostained with Anti-Rad51 antibody (ab63801) at 1/6,000 dilution. The lower panels were the same cells with Hoechst.

  • All lanes : Anti-Rad51 antibody (ab63801) at 1/2000 dilution

    Lane 1 : Molecular weight markers
    Lane 2 : HeLa cell extract

    Predicted band size: 37 kDa
    Observed band size: 37 kDa

  • Immunofluorescent detection of Rad51 foci formation induced by DNA damage. Normal human diploid cells were irradiated by X-ray (0.5 Gly) and after incubation of 6 hr, the cells were fixed and immuno-stained by using ab63801 (x100 dilution) as the primary antibody and Alexa594 labeled anti-rabbit antibody as the secondary antibody.

References

This product has been referenced in:
  • Popp I  et al. Fanconi anemia with sun-sensitivity caused by a Xeroderma pigmentosum-associated missense mutation in XPF. BMC Med Genet 19:7 (2018). Read more (PubMed: 29325523) »
  • Margalef P  et al. Stabilization of Reversed Replication Forks by Telomerase Drives Telomere Catastrophe. Cell 172:439-453.e14 (2018). Read more (PubMed: 29290468) »
See all 44 Publications for this product

Customer reviews and Q&As

1-10 of 14 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (H520 lung adenocarcinoma)
Gel Running Conditions
Reduced Denaturing (8%)
Loading amount
30 µg
Specification
H520 lung adenocarcinoma
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3%

Abcam user community

Verified customer

Submitted Nov 17 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (H1944 lung adenocarcinoma)
Permeabilization
Yes - CSK buffer
Specification
H1944 lung adenocarcinoma
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Nov 12 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (293T)
Permeabilization
Yes - 0.1% triton X100
Specification
293T
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Oct 10 2017

Application
Western blot
Sample
Human Cell lysate - whole cell (293T)
Gel Running Conditions
Non-reduced Denaturing (12%)
Loading amount
5 µg
Specification
293T
Blocking step
Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Oct 10 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Dictyostelium discoideum Cell (AX3)
Permeabilization
Yes - Triton X100
Specification
AX3
Fixative
Methanol

Abcam user community

Verified customer

Submitted Feb 26 2016

Answer

These bands could be result of a few factors. First is that the antibody is detecting a second isoform of Rad51 with similar size which is present in the nucleus. but not in the cytoplasm. The SwissProt page for this target does list 4 isoforms (http://www.uniprot.org/uniprot/Q06609). A second possibilty is digestion of your nuclear sample by proteases. We recommend addition of fresh protease inhibitors to the lysis buffer directly before use and performing the lysis on ice. A third possibility if that the target has been post translationally modified. This target can be phosphorylated. We recommend reducing and denaturing the sample by boiling in a sample buffer containing a reducing agent for 5-10 minutes. However you may wish to heat the sample for longer at 72C for up to 20 minutes.

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Question
Answer

Thank you for contacting us.

The following protocol was used with ab104306 for ICC/IF:

Preparation of slides:

1. Grow cultured cells on sterile glass cover slips at 37°C overnight.

2. Wash sample with PBS twice.

3. Fix cells for 15 minutes with 2 mL of 4% paraformaldehyde solution (pH 7.4 with NaOH in PBS).

Step-by-step procedure:

1. Permeabilize cells by incubating for 15 minutes on ice with 2 mL of 0.1% Triton X-100 in PBS.

2. Wash cells 3 times with PBS.

3. Incubate cells for 1 hour with normal goat blocking serum (1:20 in PBS).

4. Introduce primary antibodies (in appropriate dilutions) to the sample.

5. Incubate for 4 hours at room temperature or at 4°C overnight.

6. Wash with PBS for 5 minutes. Repeat 5 times.

7. Incubate cover slips in fluorescein-conjugated secondary antibodies in 5% normal blocking serum in PBS in a dark humidity chamber at 4°C for 1 hour.

(Perform all subsequent washes under dim and ambient light source.)

8. Wash sample thoroughly with PBS. Each wash lasting 5 minutes. Repeat 6 times.

9. Counter stain sample with DAPI at room temperature for 30 minutes.

10. Wash sample with PBS. Each wash lasting for 2 minutes. Repeat 3 times.

11. Mount sample by inverting them onto mounting medium on glass slides.

12. Store slides in dark at 4°C.


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

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Question



No signal. After irradiation (8Gy) he couldn’t confirm Rad51 foci formation induced by DNA damage in the nucleus. Whereas he confirmed gamma H2AX in the same condition and used same secondary antibody.
1) Abcam product code
ab63801
2) Abcam order reference number or product batch number

2-1) storage temperature of antibody
4℃
3) Description of the problem
After irradiation, Rad 51 was not stained at Double strand breaks of chromosome DNA,
but cytoplasm.
4) Sample preparation:
Species Human
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed
paraffin embedded sections, cells in culture, other:
Sample preparation
Positive control HeLa Cell+Irradiation (8Gy)
Negative control HeLa Cell + No irradiation
5) Fixation step
Yes
If yes: Fixative agent Methanol and concentration 100%
Fixation time 5 minutes
Fixation temperature -20℃
6) Antigen retrieval method No
7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution
buffers? We do not need permeabilization step because of methanol fixation, but we use
antibody dilution buffer contained 0.2% Triton-X 100 and washing buffer supplemented
0.2% Triton-X 100.
Permeabilizing agent and concentration: Triton-X 100 and 0.2%
8) Blocking agent (eg BSA, serum…): BSA
Concentration 1%
Blocking time 30 minutes
Blocking temperature Room temperature
9) Endogenous peroxidases blocked? No
Endogenous biotins blocked? No
10) Primary antibody (If more than one was used, describe in “additional notes”) : Anti-Rad 51
ANTIBODY (Rabbit)
Concentration or dilution: 1:100
Diluent buffer 1%BSA/0.2% Triton X-100
Incubation time Overnight at 4℃
11) Secondary antibody: Alexa Fluor 488 (A11008)
Species: Goat
Isotype: IgG
Reacts against: Rabbit
Concentration or dilution 1:500
Diluent buffer 1%BSA/0.2% Triton X-100
Incubation time: 1 hour
Fluorochrome or enzyme conjugate: Alexa Fluor 488
12) Washing after primary and secondary antibodies:
Buffer 0.2% Triton X-100
Number of washes 3 TIMES
13) Detection method Microscope (Fluorescence)
14) How many times have you run this staining? 3 times
Do you obtain the same results every time? Yes
Have you run a "No Primary" control?(yes/no) No
What steps have you altered to try and optimize the use of this antibody?

Read More
Answer

Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab63801 Anti-Rad51 antibody. I would also appreciate if you can confirm some further details:

1. Please be aware that non-radiated HeLa cells are not a fitting negative control, as they are given as a positive control on our datasheet. However, in this light it is even more odd that the antibody doesn't give the expected results and rather stains the actin filaments.

2.Triton can be quite harsh on some epitopes, therefore, youincrease the clarity of the staining by reducing the Triton concentration down to 0.025%, especially when you incubate over night.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

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Answer

I am happy to help. Best of luck in your experiments!

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Answer

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement for one vial of ab63801.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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1-10 of 14 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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