Overview

  • Product name
  • Description
    Rabbit polyclonal to SAP155
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Xenopus laevis
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 250 - 350 of Human SAP155.

    Read Abcam's proprietary immunogen policy (Peptide available as ab39577)

  • Positive control
    • ab39578 gave a positive result in the following lysates: Hela, Jurkat and Jurkat Nuclear. This antibody gave a positive result in IHC in the following FFPE tissue: Human pancreatic adenocarcinoma.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Preservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab39578 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 Use a concentration of 1 µg/ml. Detects a band of approximately 150 kDa (predicted molecular weight: 150 kDa).
IHC-P Use a concentration of 1 µg/ml.
ICC/IF Use a concentration of 1 µg/ml.

Target

  • Function
    Subunit of the splicing factor SF3B required for 'A' complex assembly formed by the stable binding of U2 snRNP to the branchpoint sequence (BPS) in pre-mRNA. Sequence independent binding of SF3A/SF3B complex upstream of the branch site is essential, it may anchor U2 snRNP to the pre-mRNA. May also be involved in the assembly of the 'E' complex. Belongs also to the minor U12-dependent spliceosome, which is involved in the splicing of rare class of nuclear pre-mRNA intron.
  • Sequence similarities
    Belongs to the SF3B1 family.
    Contains 11 HEAT repeats.
  • Post-translational
    modifications
    Phosphorylated. Phosphorylation occurs concomitantly with the splicing catalytic steps. Phosphorylation on Thr-244, Thr-248 and Thr-313 by cyclin-dependent kinases promotes interaction with PPP1R8 during mitosis.
  • Cellular localization
    Nucleus speckle. During mitosis, transiently dispersed from the nuclear speckles to the cytoplasm.
  • Information by UniProt
  • Database links
  • Alternative names
    • Hsh155 antibody
    • OTTHUMP00000205700 antibody
    • OTTHUMP00000205702 antibody
    • OTTHUMP00000225001 antibody
    • OTTHUMP00000225002 antibody
    • Pre mRNA processing 10 antibody
    • Pre mRNA splicing factor SF3b, 155 kDa subunit antibody
    • Pre-mRNA splicing factor SF3b 155 kDa subunit antibody
    • Pre-mRNA-splicing factor SF3b 155 kDa subunit antibody
    • PRP10 antibody
    • PRPF10 antibody
    • SAP 155 antibody
    • SAP155 antibody
    • sf3b1 antibody
    • SF3B1_HUMAN antibody
    • SF3b155 antibody
    • Spliceosome associated protein 155 antibody
    • Spliceosome-associated protein 155 antibody
    • Splicing factor 3B subunit 1 antibody
    • Splicing factor 3b, subunit 1, 155kDa antibody
    see all

Images

  • All lanes : Anti-SAP155 antibody (ab39578) at 1 µg/ml

    Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
    Lane 2 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
    Lane 3 : Jurkat nuclear extract lysate (ab14844)

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 150 kDa
    Observed band size: 150 kDa

  • ICC/IF image of ab39578 stained human HeLa cells. The cells were methanol fixed (5 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab39578, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
  • IHC image of SAP155 staining in Human pancreatic adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab39578, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

References

This product has been referenced in:
  • Eaton JD  et al. Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity. Genes Dev 32:127-139 (2018). Read more (PubMed: 29432121) »
  • Llorian M  et al. The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators. Nucleic Acids Res 44:8933-8950 (2016). WB ; Mouse . Read more (PubMed: 27317697) »
See all 4 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Answer

Thank you for contacting us.  The primary UniProt entry for this product is O75533 (this entry is reviewed).  The other two UniProt entries refer to fragments of the same protein.  We expect SAP155 to run at 146 kDa, but is possible that an additional band may be observed just above 146 kDa corresponding to the heavily phosphorylated form of the protein.  It appears that all of the SAP155 present in the HeLa cells is fully phosphorylated.  We are unsure of the identity of the weaker lower band, but it appears to be nonspecific.  I hope this helps, please let me know if you need any additional information.   

Read More

Answer

Thank you for your reply and for sending the customer's enquiry.

It may be possible to treat the lysate with a more concentrated CIP solution, however we have found that the recommended concentration tends to give the best results. I searched through some literature to see if I could find a reference of someone using concentrated CIP, but I could not find any definitive answer. I would recommend treating the samples with CIP at the recommended 1 ug protein in 10 uL of buffer, and then centrifuging the samples and aspirating some of the buffer in order to concentrate the samples before adding Laemmli buffer and loading on the gel.

I hope this will be useful, but please let me know if the customer has any further questions and I'll be happy to help. Have a great day!

Read More

Answer

Thank you for contacting us with your customer's questions. I'm hoping that I can provide some helpful responses.

1) Regarding the lysate treatment, I think it would be fine to freeze the samples prior to CIP treatment. Just lyse the cells as usual (but with a lysis buffer that does not contain sodium orthovanadate or EDTA) and freeze the lysate. Thaw when ready and proceed with the de-phosphorylation protocol.

2) Itseems that the secondpart of theprotocolis missing a few steps, so I apologize for this error.I recommend adding1-2% CIPto the CIP bufferand incubating the membrane overnightat 37 degrees C. After this incubation,wash the membrane threetimes, then proceed with theWestern blot protocol (starting with the proteinblock step).

I hope this information will be useful, but please let me know if there are any further questions and I'll be happy to help.

Read More

Answer

Thanks for getting back to me, and I'm very glad to hear that the customer was satisfied with the previous suggestions.

Every phosphatase will phosphorylate specific amino acids in proteins, and PP1 and PP2A are specific serine/threonine phosphatases. The SAP155 proteinis known to be phosphorylated at several serine and threonine residues (http://www.uniprot.org/uniprot/O75533), so PP1 and PP2A should be good phosphatases to use with this protein.

Unfortunately we don't carry phosphatases in our catalog at this time, so I will suggest searching on http://www.biocompare.com for a vendor who can provide these. Treatment can probably be applied directly to the cells in culture or to the cell lysate, but I am not sure of this and will also recommend checking with the phosphatase vendor for a complete protocol.

PMSF is an inhibitor of serine and cysteine proteases, and sodium orthovanadate is an inhibitor only of tyrosine phosphatases, so these are probably not the most relevant for the customer's study. I would suggest sodium fluoride, which is an inhibitor of serine/threonine phosphatases, or a phosphatase inhibitor cocktail which is available from other vendors as well.

I hope this information will be helpful to the customer, but please let me know if you have any additional questions and I'll be happy to help!

Read More

Answer

Thank you for your reply. My colleague Jackie is out of the office but I am happy to help you with this case. Unfortunately we have not done any further characterization of the bands that appear in the Jurkat nuclear lysate on our website. There are several known phosphorylation sites for SAP155, and the doublet in the image that you sent likely represents different phosphorylation states- http://www.uniprot.org/uniprot/O75533 I agree with Jackie that the lowest band is most likely non-specific, as there are some very faint non-specific bands on the blot on our website (though at different molecular weights than the lowest band on your blot). What kind of phosphorylation characterization studies has the customer performed? If the bands are due to phosphorylation, then sufficient treatment with phosphatases should remove the extra bands. If the customer is unhappy with the results, we will of course replace or credit/refund the original purchase. Please let me know if you have any further questions or if there is anything else that we can do for you.

Read More

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

Sign up