Overview

  • Product name
  • Description
    Rabbit polyclonal to Drosha
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Synthetic peptide, corresponding to a region between residue 1050 and 1100 of human Drosha (NP_037367.3)

  • Positive control
    • Human Breast Carcinoma, Ovarian Carcinoma, Prostate Carcinoma tissues and Mouse Teratoma tissue.
  • General notes

    Concentration is optimized for IHC and not determined

Properties

Applications

Our Abpromise guarantee covers the use of ab85027 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 1/100 - 1/500.
WB 1/500. Predicted molecular weight: 159 kDa.
ICC/IF Use a concentration of 5 µg/ml.

Target

  • Function
    Ribonuclease III double-stranded (ds) RNA-specific endoribonuclease that is involved in the initial step of microRNA (miRNA) biogenesis. Component of the microprocessor complex that is required to process primary miRNA transcripts (pri-miRNAs) to release precursor miRNA (pre-miRNA) in the nucleus. Within the microprocessor complex, DROSHA cleaves the 3' and 5' strands of a stem-loop in pri-miRNAs (processing center 11 bp from the dsRNA-ssRNA junction) to release hairpin-shaped pre-miRNAs that are subsequently cut by the cytoplasmic DICER to generate mature miRNAs. Involved also in pre-rRNA processing. Cleaves double-strand RNA and does not cleave single-strand RNA. Involved in the formation of GW bodies.
  • Tissue specificity
    Ubiquitous.
  • Sequence similarities
    Contains 1 DRBM (double-stranded RNA-binding) domain.
    Contains 2 RNase III domains.
  • Domain
    The 2 RNase III domains form an intramolecular dimer where the domain 1 cuts the 3'strand while the domain 2 cleaves the 5'strand of pri-miRNAs, independently of each other.
  • Cellular localization
    Nucleus. Nucleus > nucleolus. A fraction is translocated to the nucleolus during the S phase of the cell cycle. Localized in GW bodies (GWBs), also known as P-bodies.
  • Information by UniProt
  • Database links
  • Alternative names
    • DROSHA antibody
    • Drosha double stranded RNA specific endoribonuclease antibody
    • Drosha ribonuclease type III antibody
    • Etohi2 antibody
    • HSA242976 antibody
    • Nuclear RNase III Drosha antibody
    • p241 antibody
    • Protein Drosha antibody
    • Putative protein p241 which interacts with transcription factor Sp1 antibody
    • Putative ribonuclease III antibody
    • RANSE3L antibody
    • Ribonuclease 3 antibody
    • Ribonuclease III antibody
    • Ribonuclease III nuclear antibody
    • Ribonuclease type III nuclear antibody
    • RibonucleaseIII antibody
    • RN 3 antibody
    • RN3 antibody
    • RNase 3 antibody
    • RNase III antibody
    • RNase3 antibody
    • RNASE3L antibody
    • RNaseIII antibody
    • RNASEN antibody
    • RNC_HUMAN antibody
    see all

Images

  • All lanes : Anti-Drosha antibody (ab85027) at 1/500 dilution

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 159 kDa
    Observed band size: 145 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 63 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 8 minutes
  • ab85027 at 1/250 dilution staining Drosha in human ovarian tumor by Immunohistochemistry, Formalin-fixed, Paraffin-embedded tissue. Detection: DAB staining.
  • ab85027 at 1/250 dilution staining Drosha in mouse teratoma by Immunohistochemistry, Formalin-fixed, Paraffin-embedded tissue. Detection: DAB staining.
  • ab85027 at 1/250 dilution staining Drosha in human ovarian tumor by Immunohistochemistry, Formalin-fixed, Paraffin-embedded tissue. Detection: DAB staining.
  • ICC/IF image of ab85027 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 (ab85027, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 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:
  • Antoniali G  et al. Mammalian APE1 controls miRNA processing and its interactome is linked to cancer RNA metabolism. Nat Commun 8:797 (2017). Read more (PubMed: 28986522) »
  • Lønvik K  et al. Prognostic value of the MicroRNA regulators Dicer and Drosha in non-small-cell lung cancer: co-expression of Drosha and miR-126 predicts poor survival. BMC Clin Pathol 14:45 (2014). Read more (PubMed: 25525410) »
See all 2 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Question

1. Order details: Antibody storage conditions (temperature/reconstitution etc) The antibody stores at 4℃ as introduction.
2. Please describe the problem (high background, no staining etc). The protein drosha which should be mostly expressed in nuclear , however , it was detected mostly in cytoplasm by IHC , what’s more , it was also detected diffusedly expressed in both nuclear and cytoplasm using IF and ICC both in MDA-MB-468 and MCF-10A cell lines. .And the expression is very weak in tissues and cell lines in IF,ICC and IHC tests.
3. On what material are you testing the antibody in IHC The antibody in IHC was tested on gastric tissues. And I did the IF and ICC on both MDA-MB-468 and MCF-10A cell lines.
4. How did you fix the samples? The gastric tissues which come from gastric cancer patients were fixed in 4% paraformaldehyde at room temperature about 25℃ for 24 hours The cells which comes from both MDA-MB-468 and MCF-10A were fixed in 4% paraformaldehyde at room temperature about 25℃ for 20 minutes
5. For formalin-fixed paraffin embedded tissue: did you apply antigen retrieval step? - The gastric tissues which were fixed by paraformaldehyde were applied antigen retrieval step by heat retrieval..
6. Blocking steps:
- For HRP detection method: did you block endogenous peroxidases: (yes)
- How did you block the unspecific binding sites: serum
- For how long: 30minutes
7. Primary antibody
- At what dilution(s) have you tested this antibody(1100,1:200,1:500,1:800)
- What dilution buffer was used: PBS
- Incubation time: overnight (about 14hours)
- Incubation temperature: 4℃
- What washing steps were done: The slides were washed by PBS (pH7.2-7.6) for 5 minutes one time three times
8. Secondary antibody
- Specification (in which species was it raised against):the goat raised against rabbit - At what dilution(s) have you tested this antibody: 1:100
- What dilution buffer was used: serum
- Incubation time: 30minutes
- Incubation temperature: 37℃
- What washing steps were done: The slides were washed by PBS (pH7.2-7.6) for 5 minutes one time three times
- Do you know whether the problems you are experiencing come from the secondary? I did do some tests to exclude the problems comes from the secondary antibody in breast tissues.
9. What detection method are you using? I used thee microscope to detect IHC and ICC results and fluorescence to detect IF results.
10. Background staining See supplement figure 1.
11. Which detection system did you use? No detection system was used.
12. Did you apply positive and negative controls along with the samples? For IHC tests , I apply negative controls along with the samples but I did not apply positive controls .But I used the breast tissue as the controls to exclude the problems comes from others For ICC and IF tests , I apply both positive and negative controls along with the samples .
13. Optimization attempts
- How many times have you tried the IHC? Five times for IHC . Three times for ICC and IF.
- Do you obtain the same results every time? YES.
- What steps have you altered? 1 Changed cell lines or gastric tissues . 2 Changed the dilutions of primary antibody at1:100,1:200,1:500 and1:800 .

Read More
Answer

Thank you for contacting us. I am sorry that ab85027 is detecting an incorrect localization of Drosha in IHC. I agree that your protocol should be producing a clearer nuclear stain with less background. For our records, did you run a no-primary antibody control to rule out other sources of background? I would be happy to offer you either a free of charge replacement with an alternative antibody such as ab12286 or a credit or refund. Please let me know how you would like to proceed and I will be happy to help you further.

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Answer

Thank you for your recent contact and your kind reminder.
I have checked our internal system which stores all the in-coming and out-going messages. According to our CRM system, your enquiry was received in February (Tue 21 Feb 12) and I replied to your enquiry on the same day Tue 21 Feb 12.
- Could you please confirm the batch number and the Abcam Order Number which relates to this vial?
- Could you please also verify if you purchased this product directly from Abcam or via our Chinese distributor?
If this is the case, I would recommend you to contact our HK office directly. We have a dedicated team there and you can discuss this enquiry with my colleagues who will be happy to help you. E-mail: hk.technical@abcam.com
If you need any further assistance in the future, please do not hesitate to contact us.

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Answer

Thank you for your response.
ICC stands for for immuno-cytochemistry which requires cells (not tissues); whilst IHC is the abbreviation for immuno-histochemistry.
If you wish to use tissue sections of human gastric samples then you need an antibody which is suitable for immuno-histochemistry (IHC). I assume the samples are fixed in formalin and embedded in paraffin so you are looking for antibodies particularly for IHC-P? I can confirm that ab58589 and ab85027 both have been characterized for this type of immunoassay.
Please take a look at the on-line product datasheets to see which one would suit your need best. Your choice/decision may depend on the detection system/ sample type you wish to apply etc.
I hope this helps and if I can assist further, please do not hesitate to contact me.

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Answer

Thank you for your response.
Both antibodies (ab58589 and ab85027) are suitable for IHC-P application and they can detect human Drosha. I would suggest taking a look at the on-line product datasheets to see which one would suit your need best. Your choice/decision may depend on the detection system/ sample type you wish to apply etc.
I hope this helps and if I can assist further, please do not hesitate to contact me.

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Answer

Thank you for your enquiry regarding ab12286.
Currently, we have 12 specific publications on this product (ab12286) and the list of these papers can be viewed on the on the on-line datasheet. Since ab12286 has been tested in ICC/IF, IP, WB, ICC, ChIP; most of these articles are WB-related. We do not have any data/publication which would support its suitability for IHC since it has not been characterized yet.
The other two antibodies against Drosha (ab58589 and ab85027) have been tested in IHC-P and we guarantee them in this application but not aware of any IHC-specific published work yet. As soon as we have further information, we will update the datasheets accordingly.
I hope this helps and if I can assist further, please do not hesitate to contact me.

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Question
Answer

Thank you for your enquiry. Currently, we only sell polyclonal antibodies against Drosha. Is there any specific reason why you are interested in monoclonal rabbit antibodies? We have 2 rabbit polyclonal antibodies: ab12286 and ab85027 and one goat polyclonal antibody ab58589. If you need any further assistance in the future, please do not hesitate to contact me.

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Question
Answer

We do not give the precise concentration of IgG in this product. ab85027 is prepared to provide specific IHC staining within the recommended range of dilutions.  The concentration of IgG is less than or equal to 1 mg/ml.  For your purposes where you wish to run a normal rabbit IgG as a control, assuming 1 mg/ml for the concentration of IgG in the IHC antibody is reasonable.  I hope this is helpful. Please contact us again if you have any further questions.

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Question
Answer

Thank you for your patience. The antibody concentration for 0.1 ml was determined by extinction coefficient: absorbance at 280 nm of 1.4 equals 1.0 mg of IgG, thus the concentration would be 10 mg/ml. I am confirming this still with the lab. In the meantime, please do not hesitate to contact us if you need any more advice or information.

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