Overview

  • Product name
    Anti-GBP3 antibody
  • Description
    Rabbit polyclonal to GBP3
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant fragment containing a sequence corresponding to a region within amino acids 61-287 of Human GBP3 (NP_060754).

  • Positive control
    • Hela whole cell lysate; NCIN87 xenograft; A431, HepG2 and Molt-4 cell lysates

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.00
    Preservative: 0.01% Thimerosal (merthiolate)
    Constituents: 1.21% Tris, 0.75% Glycine, 10% Glycerol
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab97935 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 - 1/3000. Predicted molecular weight: 68 kDa.
IHC-P 1/100 - 1/500.

Target

  • Function
    Binds GTP, GDP and GMP.
  • Sequence similarities
    Belongs to the GBP family.
  • Cellular localization
    Membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • DKFZp686E0974 antibody
    • DKFZp686L15228 antibody
    • FLJ10961 antibody
    • GBP 3 antibody
    • GBP-3 antibody
    • GBP3 antibody
    • GBP3_HUMAN antibody
    • GTP binding protein 3 antibody
    • GTP-binding protein 3 antibody
    • Guanine nucleotide binding protein 3 antibody
    • Guanine nucleotide-binding protein 3 antibody
    • Guanylate binding protein 3 antibody
    • Guanylate-binding protein 3 antibody
    • guanylate-binding protein 3 delta C antibody
    • SPG3A antibody
    see all

Images

  • Anti-GBP3 antibody (ab97935) at 1/1000 dilution + Hela whole cell lysate at 30 µg

    Predicted band size: 68 kDa



    7.5% SDS PAGE
  • Immunohistochemical analysis of GBP3 in paraffin-embedded NCIN87 xenograft, using ab97935 at 1/100 dilution.

References

ab97935 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 email and for sending me the WB data which is of great help. I have not yet heard back from the lab regarding the 3 bands in the WB image for ab104293. Both antibodies do seem to give high background as well. I am wondering if a higher dilution, especially for the GBP3 antibody might help reducing the background as well as removing the extra band at 40 kDa (e.g. 1/1000). As for the GBP7 antibody, it does not seem to detect the correct band. How abundant do you think the protein might be in these samples? Maybe increasing the protein amount to up 100 ug could help, if the protein is very low abundant? Thank you so much for trying to troubleshoot. Please let me know what your results are. Should none of this help to improve the data, I'd be happy to offer a free of charge replacement, a credit or refund for these 2 antibodies. I wish you good luck, and look forward to hear back from you and assist you further.

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Answer

Thank you for contacting us. I am sorry to hear that 2 of our antibodies are giving you problems. For ab104293, regarding the 3 bands in the WB image, I will have to ask the lab for more information. For the WB protocol please see below. At which MW do you see the weak band? According to the UniProt database (http://www.uniprot.org/uniprot/Q8N8V2), no additional isoform or splice variant has been reported. For ab97935, I am not sure why you see a second band at 40 kDa. Again, according to the UniProt database (http://www.uniprot.org/uniprot/Q9H0R5), no additional isoform or splice variant has been reported. For both antibodies, could you please let me know: 1) How much protein are you loading? 2) What antibody dilutions are you using? 3) What is the blocking reagent? 4) Is the secondary antibody working well with other primaries? 5) Have you tried any other samples besides THP-1 (e.g. a positive control such as Hela or HepG2 cells)? If so, what were the results there? Western Blotting Protocol for ab104293: A. Preparation of cell lysates 1. Collect cells (confluent T-25) by trypsinization and spin. 2. Lyze the pellet with 100 µl lysis buffer on ice for 10 min. For 500,000 cells, lyze with 20 µl. 3. Spin at 14,000 rpm (16,000 g) in microfuge for 10 min at 4°C. 4. Transfer the supernatant to a new tube and discard the pellet. 5. Determine the protein concentration (Bradford assay, A280, or BCA). 6. Take x µl (= y µg protein) and mix with x µl of 2x sample buffer. 7. Boil for 5 min and cool at RT for 5 min. 8. Flash spin to bring down condensation prior to loading gel. B. Polyacrylamide gel (14.5 cm x 16.5 cm) 1. Agarose plug: 1% agarose dissolved in 1x Resolving gel buffer. 2. Resolving gel: 24 ml of a 9% gel, 5.4 ml 40% acrylamide/bisacrylamide (29:1 mix), 3 ml 8x Resolving gel buffer, 15.6 ml water, 12 µl TEMED, 60 µl 20% ammonium persulfate 3. Stacking gel: 8 ml = 1 ml 40% acrylamide/bisacrylamide (29:1 mix), 2 ml 4x Stacking gel buffer, 5 ml water, 8 µl TEMED, 21.6 µl 20% ammonium persulfate C. Preparation of gel 1. Assemble the glass plates and spacers (1.5 mm thick). 2. Pour an agarose plug (1-2 mm). 3. Pour the running gel to about 1 cm below the wells of the comb (~20 ml). 4. Seal with 1 ml water-saturated 1-butanol. 5. When gel has set, pour off the butanol and rinse with deionized water. 6. Pour the stacking gel (~5 ml) and insert the comb immediately. 7. When the stacking gel has set, place in gel rig and immerse in buffer. 8. Prior to running the gel, flush the wells out thoroughly with running buffer. D. Running the gel 1. After flash spinning the samples, load into the wells. 2. Be sure to use markers. 3. Run with constant current (35-37 mA with voltage set at > 150 V). 4. Usual running time is about 1.3 hr. E. Preparation of membrane 1. Cut a piece of PVDF membrane. 2. Wet in methanol on a rocker at RT for 5 mins. Remove methanol and add 1x Transfer buffer until ready to use. F. Membrane transfer 1. Assemble "sandwich" for Bio-Rad's Transblot. 2. Prewet the sponges, filter papers (slightly bigger than gel) in 1x Blotting buffer. Sponge - filter paper - gel - membrane - filter paper - sponge 3. Transfer for 1 hr at 15 volts at 4°C on a stir plate. Bigger proteins might take longer to transfer. For the Mini-Transblot, it's 100 V for 1 hr with the cold pack and prechilled buffer. 4. Immerse membrane in Amido-Black stain 5 mins. 5. Destain 4 x 5 mins with destaining buffer. 6. When finished, immerse membrane in Blocking buffer and block for one hour at room temperature. G. Antibodies and detection 1. Incubate with primary antibody diluted to 2 ug/ml in total volume of 3 mL in Blocking buffer for one hour at room temperature. 2. Wash 4 x 5 min with 0.05% Tween 20 in TBS. 3. Incubate with secondary antibody diluted 1:10,000 (HRP anti-rabbit) in Blocking buffer for 1 hour at room temp. 4. Wash 4 x 5 min with 0.05% Tween 20 in TBS. 5. Detect with Chemiluminescent kit. H. Stripping blot 1. Rinse blot off with 0.05% Tween 20 in PBS. 2. Put blot into Kapak bag cut to slightly bigger size than blot. 3. Add about 5 to 10 ml Stripping buffer. 4. Remove as much air as possible and seal bag. 5. Immerse into 80°C water bath and incubate for 20 min. 6. Rinse blot off with 0.05% Tween 20 in PBS. 7. Block for about 1 hr with 5% BSA/Tween 20, or overnight with 3% BSA/Tween20. Buffers for Westerns Lysis buffer: 0.15 M NaCl 5 mM EDTA, pH 8 1% Triton X100 10 mM Tris-Cl, pH 7.4 Just before using add: 1:1000 5 M DTT 1:1000 100 mM PMSF in isopropanol 1:1000 5 M e-aminocaproic acid 2x sample buffer: 130 mM Tris-Cl, pH8.0 20% (v/v) Glycerol 4.6% (w/v) SDS 0.02% Bromophenol blue 2% DTT 8x Resolving gel buffer: 100 ml 0.8 g SDS (add last) 36.3 g Trizma base (= 3 M) Adjust pH to 8.8 with concentrated HCl 4x Stacking gel buffer: 100 ml 0.4 g SDS (add last) 6.05 g Trizma base (= 0.5 M) Adjust pH to 6.8 10x Running buffer: 1 L 30.3 g Trizma base (= 0.25 M) 144 g Glycine (= 1.92 M) 10 g SDS (= 1%)--add last Do not adjust the pH!! 10x Blotting buffer: 1 L 30.3 g Trizma base (= 0.25 M) 144 g Glycine (= 1.92 M) pH should be 8.3; do not adjust To make 2 L of 1x Blotting buffer: 400 ml Methanol 200 ml 10x Blotting buffer 1400 ml water Blocking buffer: 0.5 L 3% Bovine serum albumin (Fraction V) Make up in PBS and sterile filter. Then add 0.05% Tween 20. Keep at 4°C to prevent bacterial contamination. Stripping buffer: 0.5 L (sterile filter solution and keep at 4°C) 0.2 M Glycine, pH 2.5 0.05% Tween 20 I look forward to hear back from you.

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