Astana Biomed Group, an authorized Abcam distributor in Central Asia

Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free (ab255288)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR22598-212] to TNF alpha - BSA and Azide free
  • Suitable for: WB, Flow Cyt, IP
  • Knockout validated
  • Reacts with: Mouse, Human

Overview

  • Product name

    Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free
    See all TNF alpha primary antibodies

  • Description

    Rabbit monoclonal [EPR22598-212] to TNF alpha - BSA and Azide free

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, Flow Cyt, IPmore details
    Unsuitable for: ICC/IF or IHC-P

  • Species reactivity

    Reacts with: Mouse, Human

  • Immunogen

    Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: THP-1 (treated with 80nM TPA / 100 ng/ml lipopolysaccharide / 300ng/ml Brefeldin A) and RAW 264.7 (treated with 100 ng/ml lipopolysaccharide / 300 ng/ml Brefeldin A) whole cell lysates. IP:THP-1 (treated with 80nM TPA / 100 ng/ml lipopolysaccharide / 300ng/ml Brefeldin A) whole cell lysate. Flow cyt: Human PBMC (treated with 50 ng/ml PMA/ 250 ng/ml ionomycon / 300 ng/ml Brefeldin A).

  • General notes

    Ab255288 is the carrier-free version of ab255275. This format is designed for use in antibody labeling, including fluorochromes, metal isotopes, oligonucleotides, enzymes.

     

    Our carrier-free formats are supplied in a buffer free of BSA, sodium azide and glycerol for higher conjugation efficiency.

    Use our conjugation kits  for antibody conjugates that are ready-to-use in as little as 20 minutes with

    ab255288 is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm.

    Maxpar® is a trademark of Fluidigm Canada Inc.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

Images

  • Western blot - Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free (ab255288)
    Western blot - Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free (ab255288)
    All lanes : Anti-TNF alpha antibody [EPR22598-212] (ab255275) at 1/1000 dilution

    Lane 1 : Wild-type THP-1 Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
    Lane 2 : Wild-type THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
    Lane 3 : TNF alpha knockout THP-1 Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
    Lane 4 : TNF alpha knockout THP-1 LPS treated (100 ng/ml, 16 h) and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
    Lane 5 : U937 PMA treated (10 mM, 2 days) plus 16 h no treatment and Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate
    Lane 6 : U937 PMA treated (10 mM, 2 days) and LPS treated (1 µg/ml, 16 h) plus Brefeldin A (ab120299) treated (5 µg/ml, 4 h) cell lysate

    Lysates/proteins at 30 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 25 kDa
    Observed band size: 26 kDa
    why is the actual band size different from the predicted?



    This data was developed using the same antibody clone in a different buffer formulation (ab255275).

    Lanes 1 - 6: Merged signal (red and green). Green - ab255275 observed at 26 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

    ab255275 was shown to react with TNF alpha in THP-1 wild-type cells in Western blot with loss of signal observed in TNF knockout sample. Wild-type and TNF knockout THP-1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab255275 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

  • Flow Cytometry - Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free (ab255288)
    Flow Cytometry - Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free (ab255288)

    Flow cytometric analysis of 2% paraformaldehyde-fixed, 0.1% Tween 20 permeabilized Human peripheral blood mononuclear cell (PBMC) treated with 50ng/ml PMA, 250ng/ml ionomycin and 300ng/ml Brefeldin A for 16h, labeling TNF alpha with ab255275 at 1/700 dilution (Right) compared with Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Left).

    Goat Anti-Rabbit IgG Fc (Alexa Fluor® 488) preadsorbed (ab150097), at 1/5000 dilution was used as the secondary antibody.

    Cells were surface stained with anti-CD3 conjugated to Alexa Fluor® 647. Then fixed with 2% PFA for 10min and intracellularly stained with rabbit IgG (Left) or ab236836 (Right).

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (ab255275).

     

  • Immunoprecipitation - Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free (ab255288)
    Immunoprecipitation - Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free (ab255288)

    TNF alpha was immunoprecipitated from 0.35 mg of THP-1 (human monocytic leukemia cell line) (treated with 80nM TPA overnight, replaced the culture medium with 100 ng/ml Lipopolysaccharides (LPS) for 6 hours with addition of 300ng/ml Brefeldin A (BFA) for the last 3 hours) whole cell lysate with ab255275 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab255275 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

    Lane 1: THP-1 (treated as above) whole cell lysate 10 μg (Input).
    Lane 2: ab255275 IP in THP-1 (treated as avove) whole cell lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255275 in THP-1 (treated as above) whole cell lysate.

    Blocking and dilution buffer and concentration: 5% NFDM/TBST.
    Exposure time: 15 seconds.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (ab255275).

  • Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free (ab255288)
    Anti-TNF alpha antibody [EPR22598-212] - BSA and Azide free (ab255288)

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