Overview

  • Product name
  • Description
    Rabbit polyclonal to PIWIL4
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Mouse
    Predicted to work with: Rat, Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 800 to the C-terminus of Mouse PIWIL4.

    Read Abcam's proprietary immunogen policy (Peptide available as ab23535.)

  • Positive control
    • Mouse testis lysate This antibody gave a positive signal in the following Formaldehyde fixed cell line: HeLa.

Properties

Applications

Our Abpromise guarantee covers the use of ab21869 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
ICC/IF Use a concentration of 5 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 98 kDa (predicted molecular weight: 98 kDa). Block with 5% BSA for 1 hour and incubate antibody in TBST for 1 hour or more.

Target

  • Function
    Plays a central role during spermatogenesis by repressing transposable elements and prevent their mobilization, which is essential for the germline integrity. Acts via the piRNA metabolic process, which mediates the repression of transposable elements during meiosis by forming complexes composed of piRNAs and Piwi proteins and govern the methylation and subsequent repression of transposons. Directly binds piRNAs, a class of 24 to 30 nucleotide RNAs that are generated by a Dicer-independent mechanism and are primarily derived from transposons and other repeated sequence elements. Associates with secondary piRNAs antisense and PIWIL2/MILI is required for such association. The piRNA process acts upstream of known mediators of DNA methylation. Participates to a piRNA amplification loop. Besides their function in transposable elements repression, piRNAs are probably involved in other processes during meiosis such as translation regulation (By similarity). May be involved in the chromatin-modifying pathway by inducing 'Lys-9' methylation of histone H3 at some loci.
  • Tissue specificity
    Expressed in testis. According to PubMed:17544373, it is ubiquitously expressed.
  • Sequence similarities
    Belongs to the argonaute family. Piwi subfamily.
    Contains 1 PAZ domain.
    Contains 1 Piwi domain.
  • Post-translational
    modifications
    Arginine methylation by PRMT5 is required for the interaction with Tudor domain-containing protein (TDRD1, TDRKH/TDRD2 and TDRD9) and subsequent localization to the meiotic nuage, also named P granule.
  • Cellular localization
    Nucleus. Cytoplasm. Probable component of the meiotic nuage, also named P granule, a germ-cell-specific organelle required to repress transposon during meiosis. PIWIL2/MILI is required for nuclear localization.
  • Information by UniProt
  • Database links
  • Alternative names
    • DKFZp686P01248 antibody
    • FLJ36156 antibody
    • HILI 2 antibody
    • HILI2 antibody
    • HIWI 2 antibody
    • HIWI2 antibody
    • Miwi 2 protein antibody
    • Miwi2 antibody
    • PIWI antibody
    • Piwi like 2 antibody
    • Piwi like 4 (Drosophila) antibody
    • Piwi like 4 antibody
    • Piwi like protein 4 antibody
    • PIWI like protein antibody
    • Piwi like RNA mediated gene silencing 4 antibody
    • Piwi-like protein 4 antibody
    • PIWIL 4 antibody
    • Piwil4 antibody
    • PIWL4_HUMAN antibody
    see all

Images

  • ICC/IF image of ab21869 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab21869 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • Nishimura T  et al. PNLDC1, mouse pre-piRNA Trimmer, is required for meiotic and post-meiotic male germ cell development. EMBO Rep 19:N/A (2018). Mouse . Read more (PubMed: 29444933) »
  • Ding D  et al. PNLDC1 is essential for piRNA 3' end trimming and transposon silencing during spermatogenesis in mice. Nat Commun 8:819 (2017). IHC-P ; Mouse . Read more (PubMed: 29018194) »
See all 4 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Mouse Tissue lysate - whole (mouse ES cells)
Total protein in input
100 µg
Immuno-precipitation step
Protein A/G
Specification
mouse ES cells

Abcam user community

Verified customer

Submitted Mar 20 2014

Application
Western blot
Loading amount
100 µg
Gel Running Conditions
Reduced Denaturing (Gradient Gel)
Sample
Mouse Tissue lysate - whole (Liver)
Specification
Liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Mar 14 2014

Answer

Thank you for your message and for providing this further information. I am sorry to hear the suggestions made have not improved the results on this occasion. I appreciate the time you have spent on these experiments and would be pleased to arrange a replacement (with a different lot number or an alternative product), or a credit note in compensation. I look forward to hearing from you with details of how you and your customer would like to proceed.

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Question

LOT NUMBER 841630 DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE •Species: Mouse •What’s cell line or tissue: GC-2, mouse spermatocyte •Cell extract or Nuclear extract: Cell extract •Purified protein or Recombinant protein: cell lysate PRIMARY ANTIBODY •Species: Rabbit •Reacts against: mouse piwil4 •At what dilution(s) have you tested this antibody: 2ul/ml •What dilution buffer was used: TBST with 0.1% BSA or 5% milk •Incubation time: O/N •Incubation temperature: 4℃ •What washing steps were done: wash 5min for three times with TBST DETECTION METHOD ECl POSITIVE AND NEGATIVE CONTROLS USED Yes, I loaded the positive control, mouse testis tissue (500ug), along with the samples. ANTIBODY STORAGE CONDITIONS -20℃ SAMPLE PREPARATION •What lysis buffer was used: RIPA buffer •What protease inhibitors were used: •What loading buffer was used: 6X SDS sample buffer •Phosphatase inhibitors: no add •Did you heat the samples: temperature and time: yes, 95℃ for 5 min AMOUNT OF PROTEIN LOADED 24ug ELECTROPHORESIS/GEL CONDITIONS •Reducing or non reducing gel: reducing gel •Reducing agent: DTT •Gel percentage : 7% TRANSFER AND BLOCKING CONDITIONS •Transfer conditions: (Type of membrane, Protein transfer verified): PVDF membrane •Buffer: TBST •Blocking agent: milk, BSA, serum, what percentage: 5% milk •Incubation time:1 hour •Incubation temperature: RT SECONDARY ANTIBODY •Species: Goat •Reacts against: Rabbit •At what dilution(s) have you tested this antibody: 0.4ug/ml •Incubation time: 1 hour •Wash steps: wash 5min for 10 times with TBST •Fluorochrome or enzyme conjugate: HRP •Do you know whether the problems you are experiencing come from the secondary? no HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Modify the transfer time and Ab dilution

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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 ab21869 : - When testing our antibodies, our lab uses 5% BSA as a blocking reagent, so I recommend switching to this instead of milk. Some antibodies give stronger, more specific signals on blots blocked with BSA instead of milk, so doing this may improve the results you are seeing, and reduce the non-specific bands. An example of the above is the western blot obtained with the Abcam GAPDH antibody ab9385 : Click here for the western blot image using ab9385 (or use the following: www.abcam.com/ab9385). 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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Question
Answer

Thank you for your enquiry. The mice for catalog number ab4027, the product used in the Western blot on the datasheet for ab21869, are adults, approximately 3 months old. Please let me know if you have any more questions.

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Question

BATCH NUMBER 156996 ORDER NUMBER 145613 DESCRIPTION OF THE PROBLEM Non-specific band, no band where Miwi2 is expected SAMPLE testis cell extract PRIMARY ANTIBODY ab21869: 0.5mg/ml, diluted in western washbuffer (see above), incubation for 2h, 5x 5min washes with western washbuffer before adding secondary ab DETECTION METHOD ECL ANTIBODY STORAGE CONDITIONS -20 ?C SAMPLE PREPARATION tissue was dissected, immediately snap frozen in liquid nitrogen and stored in -80?C for 2 days. lysis buffer: 50mM Tris HCl pH 7.4, 1% Triton X-100, 0.2% sodium deoxycholate, 0.2% SDS, 1mM PMSF, 0.25mM AEBSF, Roche complete prot. inhibitors according to the manufacturer's protocol. 300 ul lysis buffer / 5 mg tissue tissue was homogenized with a homogenizer, then incubated for 1h (4?C, on rotation) sample was centrifuged @ 12000 rpm for 20' AMOUNT OF PROTEIN LOADED different amounts loaded ELECTROPHORESIS/GEL CONDITIONS 10% denaturing SDS PAGE TRANSFER AND BLOCKING CONDITIONS transfer semi-dry on nitrocellulose membrane, 5V, 3h, buffer: towbin blocking: 10% milkpowder in western washbuffer (30mM Tris pH 7.5, 150mM NaCl, 0.25% Tween-20) SECONDARY ANTIBODY monoclonal anti-rabbit, peroxidase conjugate [another company] 1:5000, diluted in washbuffer (see above)for 1h, afterwards 5x 5min washes HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? lysis buffer (more prot inhibitors), incubation time of lysis buffer after homogenization, amount of protein loaded

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Answer

I'm sorry to hear you are having a problem with ab21869. This antibody has been tested in mouse samples and if your testis cell extract are from mice I would expect ab21869 to work well for you. I would like to suggest the following modifications to your protocol: -change the blocking buffer, concentration and incubation time; we typically recommend to try 5%BSA in TBST for 1 hour then incubate the primary antibody in TBST or TBST/BSA (it is worth trying both) and to incubate the secondary antibody in TBST only. I think the problem is likely to be due to the high concentration of the blocking agent as milk should be used at 5%. -I would also recommend using the antibody more diluted and incubate longer, for a slow targeted binding to the protein of interest (e.g. 1-2ug/ml overnight, you may find you can dilute the antibody more). We recommend using ECL+ or even more sensitive detection kits (e.g. Pierce super signal) and can I please suggest checking that your protease inhibitors and buffers are fresh and running a no primary control? If you still experience problems with those changes please do not hesitate to contact me again as the antibody is guaranteed by the Abpromise and I can offer you a replacement or refund,

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