Overview

  • Product name

  • Description

    Rabbit polyclonal to SEMA4A
  • Host species

    Rabbit
  • Specificity

    This antibody detects endogenous levels of total SEMA4A protein.
  • Tested applications

    Suitable for: IP, WB, ELISA, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
    Predicted to work with: Mouse, Rat
  • Immunogen

    A synthesized peptide derived from the internal region of human SEMA4A.

  • Positive control

    • Extracts from COS-7 and Jurkat cells. Human brain tissue sections.

Properties

Applications

Our Abpromise guarantee covers the use of ab70178 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
IP Use at an assay dependent concentration.
WB 1/500 - 1/1000. Predicted molecular weight: 84 kDa.
ELISA 1/10000.
IHC-P 1/50 - 1/100.
ICC/IF Use a concentration of 10 µg/ml.

Target

  • Function

    Inhibits axonal extension by providing local signals to specify territories inaccessible for growing axons.
  • Involvement in disease

    Defects in SEMA4A are the cause of retinitis pigmentosa type 35 (RP35) [MIM:610282]. RP leads to degeneration of retinal photoreceptor cells. Patients typically have night vision blindness and loss of midperipheral visual field. As their condition progresses, they lose their far peripheral visual field and eventually central vision as well.
    Defects in SEMA4A are the cause of cone-rod dystrophy type 10 (CORD10) [MIM:610283]. CORDs are inherited retinal dystrophies belonging to the group of pigmentary retinopathies. CORDs are characterized by retinal pigment deposits visible on fundus examination, predominantly in the macular region, and initial loss of cone photoreceptors followed by rod degeneration. This leads to decreased visual acuity and sensitivity in the central visual field, followed by loss of peripheral vision. Severe loss of vision occurs earlier than in retinitis pigmentosa.
  • Sequence similarities

    Belongs to the semaphorin family.
    Contains 1 Ig-like C2-type (immunoglobulin-like) domain.
    Contains 1 PSI domain.
    Contains 1 Sema domain.
  • Cellular localization

    Membrane.
  • Information by UniProt
  • Database links

  • Alternative names

    • CORD10 antibody
    • RP11 54H19 2 antibody
    • RP35 antibody
    • SEM4A_HUMAN antibody
    • Sema B antibody
    • Sema domain immunoglobulin domain Ig transmembrane domain TM and short cytoplasmic domain 4A antibody
    • Sema domain immunoglobulin domain Ig transmembrane domain TM and short cytoplasmic domain semaphorin 4A antibody
    • Sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) antibody
    • Sema4a antibody
    • SEMAB antibody
    • Semaphorin 4A precursor antibody
    • Semaphorin B antibody
    • Semaphorin-4A antibody
    • Semaphorin-B antibody
    • SEMB antibody
    see all

Images

  • All lanes : Anti-SEMA4A antibody (ab70178) at 1/500 dilution

    Lane 1 : Extracts from COS-7 cells
    Lane 2 : Extracts from Jurkat cells
    Lane 3 : Extracts from COS-7 cells with immunizing peptide at 10 µg

    Lysates/proteins at 30 µg per lane.

    Predicted band size: 84 kDa
    Observed band size: 84 kDa
    Additional bands at: 117 kDa, 26 kDa. We are unsure as to the identity of these extra bands.



    One other band detected greater then 117 kDa.
  • SEMA4A was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to SEMA4A and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab70178.
    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
    Band: Band: 90kDa: SEMA4A.
  • Immunohistochemistry analysis of paraffin-embedded human brain tissue using ab70178 at 1/50-1/100 dilution.
    Left image un-treated.
    Right image treated with immunizing peptide.
  • ICC/IF image of ab70178 stained SKNSH cells. The cells were 4% formaldehyde (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 (ab70178, 10µg/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:

See all 3 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

Thank you for your email. I have more information regarding the immunogen: The antibody binds to the PSI domain. Alignment of our immunogen sequence with human SEMA4D ACC# http://www.uniprot.org/uniprot/Q92854 shows a 25% identity in an 8 residue overlap. It is unlikely that the band at the higher molecular weight is SEMA4D as the homology is low. It is more likely to be the glycosylated protein. I hope this information is helpful. Just let me know if you have further questions.

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Answer

Thank you for your reply.

Kate is currently away from the office but I will try to help in your case as best I can.

Having reviewed the case I think you may be detecting the expected protein. In the very first images you shared with us it looks as if there is a band in all the 4 different lysates at around 95 kDa. There also appears to be a second band at ˜130 kDa in both the primary human melanocyte cells and SK-N-MC cell line. When you have reduced the background in your second blot, it seems the band at ˜120 kDa and ˜130 kDa has remained.

The SEMA4A protein is reported as having a sequence of ˜84 kDa, however there are also glycosylation events reported which may alter the size observed considerably. Whilst we have observed a band of ˜85 kDa in COS-7 and Jurkat cells, an additional band of ˜117 kDa was also observed. When the antibody was used in immunoprecipitation with Jurkat whole cell extract a band closer to ˜100 kDa was observed. These observed differences may be due to the glycosulation state in different samples, as well as the way in which the gel is run.

I have found reference to this in the literature:

Elizabeth P Smith et al: Expression of neuroimmune semaphorins 4A and 4D and their receptors in the lung is enhanced by allergen and vascular endothelial growth factor:

http://ukpmc.ac.uk/articles/PMC3118960//reload=0;jsessionid=Wd06PA8alA25vtiO4Rfc.0

This journal states that they have observed a band of 150 kDa as well as a band of 120 kDa corresponding to the cleaved SEMA4A product.

I would therefore think it worthwhile to repeat the experiment. The background has reduced considerably, I would consider decreasing the quantity of primary human melanocyte loaded on the gel to 5 ug lane, keeping the other cell lines at 20 ug/well.

The final blot you shared, was the jurkat cell line run? If not I would try this again too as it is a good positive control to use with the antibody.

If you would like, I can see if I can provide you with the blocking peptide to see if the bands disappear when treated with it? Or if you are still not satisfied with the results I can provide you with a replacement antibody of your choice.

I hope this information has been of some help. I look forward to receiving your reply.

Read More

Answer

Thank you for taking the time to contact us with these details. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

The details you have kindly provided will enable us to investigate this case for you and this is also helpful in our records for monitoring of quality.

I would like to reassure you that ab70178 is tested and covered by our 6 month guarantee for use in WB and human samples. In the event that a product is not functioning in the applications cited on the product data sheet, we will be pleased to provide a credit note or free of charge replacement.

Reviewing this case, I would like to offer some suggestions to help optimise the results. I would also appreciate if you can confirm some further details:

1. Please provide the order number and date of purchase

2. Please confirm which lysis buffer was used? I can recommend to try RIPA if this has not already been used. This should provide a suitable protein preparation. The sample should be reduced and denatured before running on a reducing gel.

3. I can suggest to try BSA rather than milk to block, and not to mix blocking agents through the experiment. This can sometimes help to improve results, particularly if the blot is very dark.

4. Try the antibody at a lower dilution to reduce the background, try 1::2000.

5. Is the current vial of secondary antibody working well with other primary antibodies? Try a no primary control. The concentration of secondary may need to be reduced in order to help optimize the results.

6. I can recommend to include some wash steps if this has not already been tried. Wash in PBS containing 0.2% Tween 4 times for 5 minutes at each appropriate wash step. This should help to wash away any excess antibody.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.

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