Overview

  • Product name

  • Description

    Rabbit polyclonal to UBR1
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WBmore details
  • Species reactivity

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

    Synthetic peptide corresponding to an internal region of Human UBR1 (UniProt Q8IWV7).

  • Positive control

    • HUVEC cell lysate

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer

    pH: 7.40
    Preservative: 0.02% Sodium azide
    Constituents: 49% PBS, 50% Glycerol, 0.88% Sodium chloride
    Note: PBS is without Mg2+ and Ca2+.
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Purification notes

    ab138267 was affinity purified from rabbit antiserum by immunogenic peptide affinity chromatography.
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab138267 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 1/500 - 1/1000. Predicted molecular weight: 200 kDa.

Target

  • Function

    E3 ubiquitin-protein ligase which is a component of the N-end rule pathway. Recognizes and binds to proteins bearing specific N-terminal residues that are destabilizing according to the N-end rule, leading to their ubiquitination and subsequent degradation. May be involved in pancreatic homeostasis. Binds leucine and is a negative regulator of the leucine-mTOR signaling pathway, thereby controlling cell growth.
  • Tissue specificity

    Broadly expressed, with highest levels in skeletal muscle, kidney and pancreas. Present in acinar cells of the pancreas (at protein level).
  • Pathway

    Protein modification; protein ubiquitination.
  • Involvement in disease

    Defects in UBR1 are a cause of Johanson-Blizzard syndrome (JBS) [MIM:243800]. This disorder includes congenital exocrine pancreatic insufficiency, multiple malformations such as nasal wing aplasia, and frequent mental retardation. Pancreas of individuals with JBS do not express UBR1 and show intrauterine-onset destructive pancreatitis.
  • Sequence similarities

    Belongs to the UBR1 family.
    Contains 1 RING-type zinc finger.
    Contains 1 UBR-type zinc finger.
  • Developmental stage

    Expressed in fetal pancreas.
  • Domain

    The RING-H2 zinc finger is an atypical RING finger with a His ligand in place of the fourth Cys of the classical motif.
  • Post-translational
    modifications

    Phosphorylated upon DNA damage, probably by ATM or ATR.
  • Cellular localization

    Cytoplasm > cytosol.
  • Information by UniProt
  • Database links

  • Alternative names

    • E3 ubiquitin-protein ligase UBR1 antibody
    • JBS antibody
    • N-recognin-1 antibody
    • ubiquitin protein ligase E3 component n-recognin 1 antibody
    • Ubiquitin-protein ligase E3-alpha-1 antibody
    • Ubiquitin-protein ligase E3-alpha-I antibody
    • UBR1 antibody
    • UBR1_HUMAN antibody
    see all

Images

  • Anti-UBR1 antibody (ab138267) at 1/500 dilution + HUVEC cell lysate at 30 µg

    Predicted band size: 200 kDa

References

ab138267 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Answer

Thank you for your reply.
Unfortunately I'm not terribly familiar with NP-40 substitutes. I know that Triton X-100 can be substitued for NP-40, but I don't really know what, if any, impact an NP-40 substitute would have.
The WB conditions used for this antibody are below:
Cell Lysis and WB Protocol
Cell Lysis Protocol
1. For adherent cells, wash cells with Wash Buffer (4℃) once. Add 5ml Wash Buffer (4℃) per T75 flask and immediately scrape the cells off with a rubber scraper. Collect cells by centrifugation (3000rpm, 5min) at 4℃ and remove Wash Buffer.
For suspension cells, cells were collected and washed twice with 5ml Wash Buffer (4℃) per T75 flask by centrifugation (3000rpm, 5min) at 4℃ and remove Wash Buffer.
2. Leave the tube containing cells on ice for 10 minutes. Then add Cold (4℃) Lysis Buffer to the cells (150ul per 1X106-1X107cells).
3. Mix the cells in the buffer and roll tube continuous on ice for 30 minutes at 4℃.
4. (Optional) Sonication for 4X8 at output level 5 for whole cell lysis.
5. Collect the supernatant by centrifugation (12000 rpm, 10 minutes) at 4 ℃. Assay protein concentration by BCA. The cell lysis can be stored at -70℃ or ready to use.
Wash Buffer (4℃):
10mM PBS containing 1mM NaF and 1mM Na3VO4
Whole Cell Lysis Buffer (4℃):
Solution Final Concentration
KCl 50mM
NP-40 1%
HEPES (pH7.8) 25mM
Leupeptin (SIGMA) 100ug/ml
Aprotinin (SIGMA) 20ug/ml
DTT 125uM
PMSF 1mM
Na3VO4 1mM
NaF 1mM
Western Immunoblotting Protocol
Blocking
1. Block membrane by incubating 1 hour at room temperature or overnight at 4℃ with shaking in Blocking Solution (5% BSA, 0.05% Tween-20 in TBS (50mM Tris, 100mM NaCl, pH 7.6).
Incubation with Primary Antibody
2. Dilute primary antibody at the appropriate dilution in Blocking Solution.
3. Incubate the membrane with diluted primary antibody for 1 hour at 37℃, or 2 hours at room temperature, or overnight at 4℃ with agitation.
4. Remove antibody solution. Wash the membrane 3 times for 5-10 minutes each time at room temperature in TBST (50mM Tris, 100mM NaCl, 0.05% Tween-20, pH 7.6) with shaking. Note: Increase the concentration of Tween-20 to 0.1% reduces the background and increases the specificity, but it will reduce the sensitivity.
Incubation with Second Antibody
5. Incubate membrane with secondary AP conjugate diluted (according to manufacturer’s instructions) in Blocking Solution for 1 hour at room temperature with shaking.
6. Repeat Step 4.
7. Wash membrane with TBS for 2-5 minutes before proceeding Chemiluminescent Reaction.
Chemiluminescent Reaction
8. Prepare and use the Chemiluminescent substrate according to the manufacturer’s instructions.
9. Immediately wrap the membrane and expose to X-ray films for 10 second to 1 hour period. The exposure time may vary according to the mount of antibody and antigen.
Peptide Competition
Before proceeding Western Immunoblotting, add Blocking Peptide to the diluted primary antibody in a molar ratio of 10:1 (peptide to antibody) and incubate the mixture at 4℃ for overnight or at room temperature for 2 hours.
I hope this information helps. If these suggestions do not improve the results, please let me know.

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Question

Following is information for how the antibody, anti-UBR1 ab138267, was tested.

Samples: HBEC-1, HBEC-3 cell lines (immortalized human bronchial epithelial cells)

Sample preparation: Samples were grown in 6 well plates. Samples were removed with 500 µL cellgro Trypsin-EDTA, 1X (0.05% trypsin/ 0.53 mM EDTA in HBSS) neutralized with an equal amount of ScienCell TNS. Samples were washed one time with 1X DPBS. Samples were placed in 30 – 50 µL M2 lysis buffer on rotating wheel for 30 min. Cell extracts were clarified by centrifugation at > 13,000 RPM for 5 min. Proteins were quantified with Bio-Rad Protein Assay Dye Reagent Concentrate (Bradford). 40 µg of protein in 5X loading buffer with 2-mercaptoethanol were boiled at 100°C for 4 min and then loaded on discontinuous 12% SDS-polyacrylamide gel.

Transfer method: Samples were transferred to PVFD membrane with Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell

Blocking time, temp and %: Membrane was blocked at RT in 5% nonfat-milk in PBST for 1 hour on orbital shaker.

Antibody dilutions and incubation times/ temp: Ab dilutions were 1:1000, 1:500, and 1: 250. All incubation times were approx 2 – 3 h at RT on orbital shaker with subsequent ON incubation at 4°C. Membrane was washed 3X for 10 min in PBST. 2° Ab, Rockland Anti-RABBIT IgG (H&L) (GOAT) Antibody Peroxidase Conjugated, at 1:2000 dilution was applied for 1 h. Membrane was again washed 3X for 10 min in PBST.

Detection method: Protein was detected with Millipore Immobilon Western Chemiluminescent HRP Substrate using ImageQuant LAS 4000

Attached are images of results. Two molecular markers were used. Life Technologies MagicMark XP Western Protein Standard: 220, 120, 100, 80, 60, 50, 40, 30, 20 kDa and Bio-Rad Prestained SDS-PAGE Standards Broad Range: approx. 196, 104, 59, 41, 27, 21, 16, 7 kDa.

As you can see, there are many additional bands and I was unable to see the expected 200 kDa band.

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Answer

Thank you for your reply and for providing that additional information.
Do you know what type of lysis buffer that you used? For cytosolic proteins we typically recommend NP-40.
Do you have a lower percentage gel that you could run? Typically for proteins > 100 kDa, we recommend gel percentages between 4 - 8%.
Plus, large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation. Plus lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily. But methanol is only necessary if using nitrocellulose. Since you're using PVDF, methanol can be removed from the transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich. Also you should try a wet transfer overnight at 4oC instead of semi-dry transfer if you aren't already.
Have you tried blocking in 5% BSA instead of milk? Also you may want to try blocking overnight instead of 1 hr at RT.
For the primary antibody, you may want to try a 1/2000 dilution in with the blocking agent. Also, the secondary antibody sounds a bit too concentrated at 1/2000. You may want to try 1/5000 or 1/10,000 and incubate this for 1 hr at RT. You may also want to run a no primary control to see if the secondary is binding non-specifically.
If these suggestions do not improve your results, please let me know and we can look into replacing or refunding the product for you. I look forward to your reply.

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