Goat F(ab')2 Anti-Mouse IgM mu chain (HRP) (ab5930)

Submit a review Q&A (1)References (8)

Overview

  • Product name
    Goat F(ab')2 Anti-Mouse IgM mu chain (HRP)
    See all IgM secondary antibodies
  • Host species
    Goat
  • Target species
    Mouse
  • Specificity
    Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-Peroxidase, anti-Goat Serum, Mouse IgM and Mouse Serum. No reaction was observed against anti-Pepsin or anti-Goat IgG F(c). Specificity was confirmed by ELISA at less than 1% cross reactivity against other mouse heavy or light chain isotypes.
  • Tested applications
    Suitable for: ICC/IF, IHC-Fr, Dot blot, WB, ELISA, Electron Microscopy, IHCmore details
  • Immunogen

    Mouse IgM mu chain

  • Conjugation
    HRP

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C.
  • Storage buffer
    pH: 7.2
    Preservative: 0.01% Gentamicin sulphate
    Constituents: 0.87% Sodium chloride, 1% BSA, 0.3% Potassium phosphate
  • Concentration information loading...
  • Purification notes
    This product was prepared from monospecific antiserum by immunoaffinity chromatography using Mouse IgM coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities, pepsin digestion and chromatographic separation.
  • Conjugation notes
    Horseradish Peroxidase (HRP) (Molecular Weight 40,000 daltons)
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab5930 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 1/500 - 1/5000.
IHC-Fr Use at an assay dependent dilution.
Dot blot 1/5000 - 1/100000.
WB 1/2000 - 1/10000.
ELISA 1/90000.
Electron Microscopy Use at an assay dependent dilution.
IHC 1/500 - 1/5000.

References

This product has been referenced in:
  • Xu W  et al. Emission of sulfur dioxide from polyurethane foam and respiratory health effects. Environ Pollut 242:90-97 (2018). Read more (PubMed: 29960929) »
  • Sahin A  et al. Human SOD1 ALS Mutations in a Drosophila Knock-In Model Cause Severe Phenotypes and Reveal Dosage-Sensitive Gain- and Loss-of-Function Components. Genetics 205:707-723 (2017). Read more (PubMed: 27974499) »
See all 8 Publications for this product

Publishing research using ab5930? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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Question

I finished western blotting experiment yesterday. The expression of CYP1A1 and telomerase in lung cancer cell lines were detected. There is no signal developed on the telomerase membrane. Multi-bands were developed on CYP1A1 memebrane (Please find attached pictures). The procedures are described as follows: 1) Sample protein concentration was 20 ug. Same samples were loaded indentically onto two gels. Two gels (10% precast gel) were run together. 2) Membranes transfer was successful, visualised using ponceau red. The membranes was destained completely in TBST. They were incubated separately in blocking buffer (5% non-fat milk+TBST (Bio-Rad)) overnight at 4oC with agitation. 3) Then the membranes were treated with their specific antibodies for 2 hours at 4oC with agitation (CYP1A1 1:500 in blocking buffer; Telomerase 1:500 in TBST + 1% BSA (Sigma A4503)). 4) Membranes were rinsed in TBST, repeated 3x5 minutes. 5) CYP1A1 membrane was incubated with goat anti-rabbit IgG HRP-conjugate (Upstate) (1:5000 in blocking buffer) for 80 minutes at room temperature with agitation. Telomerase membrane was incubated with goat polyclonal to mouse IgM mu-F(ab)2 Fragment (HRP) (AB5930) (1:7500 in TBST + 1% BSA) for 80 minutes at room temperature with agitation. 6) Membranes were rinsed in TBST, repeated 3x5 minutes. Bands were developed using DAB/HRP detection. In the CYP1A1 detection, the multi bands developed because the primary antibody is polyclonal, but in the HL cell line, there are more bands than the other cell lines, are they non-specific banding? Also, it seems that HL and CD cell lines have much longer bands than the others, do you think that is normal and correct? Regarding the telomerae detection, was the concentration of first and secondary antibody too low? Why the molecular weight marker (Sigma) is not visualised on this membrane but developed well on the other one (CYP1A1)? Does it because of different dilution buffer used in two detections (milk-CYP1A1 + TBST, BSA-Telomerase +TBST)? I would be very grateful if you could give me any suggestions. I lookforward to hearing from you soon. Regards,

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Answer

Thank you for your enquiry. Well done on getting your western blot to work. I have read your comments and have examined your blot images. I would like ot take each of your antibodies one at a time: ab3568 - Cytochrome P450 1A1 antibody (ab3568). You have obtained good signal using 20ug of lysate. The blot that you have e-mailed me would have fewer bands were the exposure to DAB have been reduced. We generally advise the use of chemiluminescence (ECL+). This is a more sensitive approach with which you can perform many exposures of the film to the blot. A shortened exposure would have generated significantly fewer bands. Given that you have detected bands on the blot very easily; to improve the specificity of the antibody and decrease the number of non-specific bands may I suggest that you use the antibody diluted to >1:500 e.g. 1:800 or 1:1000. This should serve to improve the recognition of the antibody for its target. I think that the reason why you are observing differences in the "length" of the bands and the number of cross reacting bands is due to the fact that the mass of the lysates that you are using are subtly different. The "longer" bands are due to diffusion of the lysates into adjacent areas and it is no consequence that there are more cross reacting bands on account of the larger mass. May I suggest that you try using the antibody diluted >1:500 and use an ECL or ECL+ detection system (provided you have a dark room), perform different exposure lengths and normalise your lysates so that you can be confident that they are all 20ug. To quantitate the mass of protein you will need to perform a Bradford assay or similar protein quant. With regards the telomerase, please can you tell me which of the antibodies that you were using e.g.abxxxx. I cannot imagine that the reason that you do not have any bands on the second blot is due to the use of BSA. BSA often gives a better, cleaner more specific blot. Can you tell me whether this blot gave a good Ponceau S staining? Were there any differences in the two blots. The transfer is the most likely problem, but if you tell me the antibody that you used I can check the datasheet to determine whether I can offer any more tips specific to the antibody e.g. dilution etc. I look forward to hearing from you with further details. I hope this information helps, please do not hesitate to contact me should you require further assistance.

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