Overview

  • Product name
    Anti-Lamin B1 antibody [119D5-F1]
    See all Lamin B1 primary antibodies
  • Description
    Mouse monoclonal [119D5-F1] to Lamin B1
  • Host species
    Mouse
  • Specificity
    Reacts with an epitope located C-terminal of residue 231 in lamin B1. Reacts against lamin B1, does not cross react with lamin B2. Lamins do not appear to be universally distributed among different cell and tissue types. ab8982 has been shown to react with HeLa and 3T3 cells in immunocytochemistry. Other cell/tissue types have not been tested.
  • Tested applications
    Suitable for: WB, Dot blot, ELISA, Flow Cyt, ICC, ICC/IF, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Sheep, Rabbit, Cow, Dog, Human
  • Immunogen

    Tissue, cells or virus corresponding to Rat Lamin B1 (C terminal). Immunogen = Purified Rat liver lamins

  • Epitope
    C-terminal to residue 231.
  • Positive control
    • NIH 3T3 whole cell lysate (ab7179) can be used as a positive control in WB. ICC/IF: Wildtype HAP1 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab8982 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 1/100 - 1/1000. Predicted molecular weight: 67 kDa.
Dot blot Use at an assay dependent concentration.
ELISA Use at an assay dependent concentration.
Flow Cyt 1/100 - 1/200.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC 1/20 - 1/50.
ICC/IF Use a concentration of 0.5 µg/ml.
IHC-Fr Use at an assay dependent concentration.

Target

  • Function
    Lamins are components of the nuclear lamina, a fibrous layer on the nucleoplasmic side of the inner nuclear membrane, which is thought to provide a framework for the nuclear envelope and may also interact with chromatin.
  • Involvement in disease
    Defects in LMNB1 are the cause of leukodystrophy demyelinating autosomal dominant adult-onset (ADLD) [MIM:169500]. ADLD is a slowly progressive and fatal demyelinating leukodystrophy, presenting in the fourth or fifth decade of life. Clinically characterized by early autonomic abnormalities, pyramidal and cerebellar dysfunction, and symmetric demyelination of the CNS. It differs from multiple sclerosis and other demyelinating disorders in that neuropathology shows preservation of oligodendroglia in the presence of subtotal demyelination and lack of astrogliosis.
  • Sequence similarities
    Belongs to the intermediate filament family.
  • Post-translational
    modifications
    B-type lamins undergo a series of modifications, such as farnesylation and phosphorylation. Increased phosphorylation of the lamins occurs before envelope disintegration and probably plays a role in regulating lamin associations.
  • Cellular localization
    Nucleus inner membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • ADLD antibody
    • lamin B1 antibody
    • Lamin-B1 antibody
    • LMN antibody
    • LMN2 antibody
    • LMNB antibody
    • Lmnb1 antibody
    • LMNB1_HUMAN antibody
    • MGC111419 antibody
    • OTTHUMP00000159218 antibody
    see all

Images

  • All lanes : Anti-Lamin B1 antibody [119D5-F1] (ab8982) at 1/2000 dilution

    Lane 1 : Whole cell lysate prepared from human mammary cells (24 hour culture)
    Lane 2 : Whole cell lysate prepared from human mammary cells (48 hour culture)

    Lysates/proteins at 50000 cells per lane.

    Secondary
    All lanes : HRP conjugated goat anti-mouse polyclonal at 1/5000 dilution

    Developed using the ECL technique.

    Predicted band size: 67 kDa
    Observed band size: 70 kDa
    why is the actual band size different from the predicted?


    Exposure time: 40 seconds

    See Abreview

  • Methanol fixed HeLa and 3T3 cells (ab7179) were stained with ab8982 (1/50 and 1/20 respectively). The cells were fixed in 100% methanol for 6 minutes at -20°C. ab8982 clearly stains the nuclear envelope with very little backgrouns staining. 

    A: HeLa cells + ab8982 (1/50) (green)
    B: HeLa cells counterstained with DAPI (blue)
    C. 3T3 cells + ab8982 (1/20) (green)
    D. 3T3 cells counterstained with DAPI (blue)

  • ab8982 staining Lamin B1 in wild-type HAP1 cells (top panel) and LMNB1 knockout HAP1 cells (bottom panel). The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab8982 at 0.5μg/ml and ab202272 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

    Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  • Overlay histogram showing HeLA cells stained with ab8982 (red line). The cells were fixed with 100% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum (ab7481) / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab8982, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was Mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

  • Immunohistochemistry on frozen sections of swine liver showing nuclear lamina staining in hepatocytes
  • Immunohistochemistry on frozen sections of human colon showing nuclear lamina staining in epithelial and connective tissue cells

References

This product has been referenced in:
  • Huang T  et al. Curcumin mitigates axonal injury and neuronal cell apoptosis through the PERK/Nrf2 signaling pathway following diffuse axonal injury. Neuroreport 29:661-677 (2018). Read more (PubMed: 29570500) »
  • Kim JK  et al. Nuclear lamin A/C harnesses the perinuclear apical actin cables to protect nuclear morphology. Nat Commun 8:2123 (2017). Read more (PubMed: 29242553) »
See all 20 Publications for this product

Customer reviews and Q&As

1-10 of 13 Abreviews or Q&A

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (Brain)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tri-Sodium Citrate
Permeabilization
Yes - with blocking
Specification
Brain
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Juliana Schneider

Verified customer

Submitted Mar 28 2017

Application
Western blot
Loading amount
15 µg
Gel Running Conditions
Non-reduced Denaturing (8%)
Sample
Human Tissue lysate - whole (Brain)
Specification
Brain
Blocking step
5% BSA + 5% Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Oct 27 2014

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1136297.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Answer

Have a lovely holiday and please ask your colleague to confirm if you wish to get a replacement and when.

I look forward to hearing from you soon.

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Answer

Thank you for getting back to me and for providing some further details.

I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.

I could offer you either a new vial as a free charge replacement or a credit note which you can use in the future. Please do let me know how you wish to proceed with your enquiry.

I look forward to hearing from you soon.

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Question

1) Abcam product code ab 8982

2) Abcam order reference number or product batch number lot GR43517-7

3) Description of the problem
I isolate nuclei and prepare nuclear protein extracts from rabbit liver. I have planned to use lamina B as my control. However, I don’t get signals from lamina B.

4) Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…) nuclear protein extracts
Lysis buffer

124 mM Tris-HCl, 20% glycerol, 4% SDS, 10% b-mercaptoethanol, 0,005% bromophenol blue, pH 6.8

Protease inhibitors: PMSF
Phosphatase inhibitors
Reducing agent
Boiling for ≥5 min? yes/no
Protein loaded ug/lane or cells/lane 30 mg/lane
Positive control
Negative control

5) Percentage of gel 4% and 10%
Type of membrane nitrocellulose
Protein transfer verified Ponceau
Blocking agent and concentration 5% milk in PBST buffer
Blocking time 2 h
Blocking temperature RT – room temperature

6) Primary antibody (If more than one was used, describe in “additional notes”) : anti lamin B
Concentration or dilution 1:200
Diluent buffer 5% milk in PBST buffer
Incubation time 14 h
Incubation temperature: 4°C

7) Secondary antibody: HAMPO
Species: horse
Reacts against: mouse
Concentration or dilution 1:10 000
Diluent buffer 5% milk in PBST buffer
Incubation time 2h
Incubation temperature: RT
Fluorochrome or enzyme conjugate: HRP

8) Washing after primary and secondary antibodies:
Buffer PBST
Number of washes 3 for 10 minutes

9) Detection method
The fluorescence signal was recorded with the Fuji Film FLA-3000 Fluorescent Image Analyzer (Raytest Isotopenmebgera¨te GmbH).
I also used Biomax Light Film (Kodak).

10) How many times have you run this staining? 2
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? I only change the detection method.


11) “additional notes”
I also used PPAR antibody (primary antibody), but I didn’t have problems with detection.

Read More
Answer

Thank you for your enquiry regarding ab8982 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this antibody.

A) Image: Both images labelled as PPAR. I am a bit confused. Could you attach an image for Lamin B1 which has the MW markers labelled properly?

B) Lysis buffer: Has the customer used RIPA buffer which contains not one - but a mixture of different proteinase inhibitors. It could be that the lower band (on the top image at around 55 kDa) represent some partially degraded protein.

C) Dilution: Has the customer checked this antibody at a dilution of 1/100 to see if the intensity of the signal changes?

I look forward to hearing from you soon.

Read More

Answer

I am sorry to hear that you have been experiencing problems using this product in the application that you wish.

All our customer feedback, including complaints are monitored weekly by our in house technical support team. If a product is at fault the technical support team will consider removing the product from our catalogue in order to avoid future customer inconvenience. We have no complaint about this batch (1216925) at all.

As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased

Could you please provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.

Thank you for your understanding and co-operation in this matter. I look forward to hearing from you soon and resolving this issue as soon as possible.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Human mammary cell)
Loading amount
50000 cells
Specification
Human mammary cell
Gel Running Conditions
Reduced Denaturing (8%)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 22 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Colon cell line)
Loading amount
20 µg
Specification
Colon cell line
Gel Running Conditions
Reduced Denaturing (4-12%)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Dec 30 2010

Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 102450 DESCRIPTION OF THE PROBLEM No Signal at around 70kD. However, weird smaller MW bands appear after long exposure of the film. SAMPLE NRK nuclear extract PRIMARY ANTIBODY I used 1:100 dilution into I-block. The second time I tried 1:50 dilution. I incubated for 1 hour at least at room temperature, then three washes with I-block. DETECTION METHOD We used CPD-Star system as our substrate for alkaline phosphatase. Expose film from 1 minute, 5 minute, 1 hour, up to overnight exposure but not seeing a signal at the correct size POSITIVE AND NEGATIVE CONTROLS USED Positive control: nuclear extract of NRK cells. This nuclear extract has been verified that it contains nuclear components by probing with anti-Nups antibody. I do not have a negative control. ANTIBODY STORAGE CONDITIONS 100ul at -20C SAMPLE PREPARATION Nuclear extract in 10%sucrose, , 0.1mM MgCl2, 10mM TEA pH 7.4 and protease inhibitors (leupeptin, Pepstatin, TAME, BAME) Samples heated at 100C with sample loading buffer for 1minute before loading to 11% polyacrylamide gel AMOUNT OF PROTEIN LOADED Serial dilution: 100ug, 50ug, 25ug and 12.5ug ELECTROPHORESIS/GEL CONDITIONS 11% denaturing SDS-PAGE TRANSFER AND BLOCKING CONDITIONS transfer overnight at 30V. blocking for 1 hour in I-block SECONDARY ANTIBODY Anti-mouse antibody conjugated with alkaline phosphatase for 30 minutes at room temperature. 3 washes afterwards with I-block HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? I increased the concentration of the antibody. Also increase the amount of proteins I loaded.

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Answer

I'm sorry to hear you are having a problem with ab8982. This antibody has been purchased many times in the past few months without any reported problems, so I am fairly confident that we can modify your protocol and get the antibody to work for you. I would like to suggest the following modifications to your protocol: 1) Please incubate in primary antibody overnight at 4 degrees to allow for a slower more specific binding and a longer time for the antibody to interact with antigen, 2) Please block for 30 minutes at room temp and incubate in secondary at room temp for one hour, 3)I am not certain of the constituents of I-block, but we often use 5% milk to block and if you are not seeing a signal you can dilute the antibodies in TBS-T instead of blocking buffer, 4) We routinely use the ECL plus detected method as this is highly sensitive and specific. I have attached the protocol that I would use with the antibody below. Western Blotting 1) To a sample of protein solution containing 1-100 ng of the target protein, add an equal volume of 2x SDS-PAGE sample buffer. For reduced samples, the sample buffer should be supplemented with DTT or 2-mercaptoethanol. For non-reduced samples, the DTT or 2-mercaptoethanol is not added (step 2 can also be left out dependent on sample). Denature the proteins by heating the sample to 95 oC, or boiling, for 5 min. 2)Load the sample onto an SDS-polyacrylamide gel and run the gel under standard conditions. 3) Transfer the proteins to a nitrocellulose or PVDF membrane using semi-dry or wet transfer methods. Please note: for PVDF it is essential to pre-wet the membrane in methanol prior to transfer. 4) If required, the efficiency of transfer can be determined by staining the membrane briefly (10 s) in Ponceau stain. The stain can be removed by washing in PBST or TBST. We would recommend not washing blots in distilled water as this can strip off proteins in some circumstances. 5) Block the membrane with a blocking buffer containing dried milk or BSA, or one from a commercial source (eg 5% w/v nonfat dry milk or 5% w/v BSA in PBST or TBST ). Incubate for 30 min at room temperature. 6)Dilute the primary antibody. We recommend incubating 1ml of primary antibody solution per 15 cm2 blot in a sealed bag, hybridisation tube or 50ml Falcon tubes can also be used (~2.5ml primary antibody/blot). Incubate overnight at 4 oC with agitation. 7) Rinse the blot in PBST or TBST and then wash twice for 5 min each and twice for 15 min at room temperature. 8)Dilute the horseradish peroxidase (HRP) labeled secondary antibody at the recommended dilution (1/5000 is usually a good working dilution although this needs to be optimised for the particular application). 9) Rinse the blot in PBST or TBST and then wash twice for 5 min each and twice for 15 min at room temperature. An optional PBS or TBS wash may be preformed as Tween may interfere with some ECL systems. 10) Perform ECL detection using the appropriate ECL solution. Please let me know if this helps and do not hesitate to contact us for further advice.

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1-10 of 13 Abreviews or Q&A

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