Recombinant Anti-GRP78 BiP antibody [EPR4040(2)] (ab108613)

Thank you for contacting us. Below is the protocol used to validate ab108613 in IHC-P. Please let me know if you need any additional information or assistance.

1. Solutions and reagents
1.1. Xylene
1.2. Ethanol, anhydrous denatured, histological grade (100%, 95%, 70%)
1.3. Washing buffer: TBST washing buffer: 1XTBS/0.1% Tween-20
To prepare stock solution of 10X TBS: add 24.2 g Trizma base and 80 g sodium chloride to 1L of dH2O.
Adjust pH to 7.6.
Working solution. 1XTBST/0.1% Tween-20: add 100ml 10XTBS to 900 ml dH2O. Add 1 ml Tween-20 and
mix well.
1.4. Distilled water (dH2O)
1.5. Antigen Retrieval Solution: 0.01M Sodium Citrate Buffer, pH 6.0
To prepare stock solutions:
Solution A. 0.1 M citric acid solution: dissolve 21.0 g of citric acid, monohydrate (C6H8O7.H2O) in 100 ml
of dH2O.
Solution B. 0.1M sodium citrate solution: dissolve 29.4 g trisodium citrate dihydrate (C6H5Na3O7.2H2O)
in 100 ml of dH2O.
Working solution: Add 9 ml of Stock solution A and 41 ml of stock solution B to 450 ml of dH2O. Adjust
pH to 6.0.
1.6. 3% Hydrogene Peroxide
1.7. Blocking buffer: PBS + 10% serum (serum origin depends on the host of the secondary antibody)
1.8. Hematoxylin QS.
1.9. Permanent Mounting medium.
2. Protocol
2.1. Deparaffinization/Rehydration
2.1.1. Heat slides in an oven at 65 °C for 1 hour.
2.1.2. De-paraffinize/hydrate using the following series of washes: two Xylene washes (5 min each), followed by two
100% ethanol rinses (5 min each), followed by 95% ethanol, 70% ethanol, 50% ethanol, 30% ethanol, followed by H
2
O
and a TBST wash for 5 min on a shaker.
2.2. Antigen Retrieval
2.2.1 Immerse slides into staining dish containing Antigen Retrieval Solution.
2.2.2 Place covered staining dish into the rice cooker. Add 120 mL dH
2
O and press “cook”.
2.2.3 When “cook” is turned to “warm” (about 20–30 min), unplug the cooker and remove the staining dish to the bench
top.
2.2.4 Allow to cool down, without cover, for 20 min.
2.3. Staining
2.3.1. Wash slides with TBST for 5 min on a shaker.
2.3.2. Inactivate endogenous peroxidase by covering tissue with 3% hydrogen peroxide for 10 min.
2.3.3. Wash slides three times with TBST (3 min each on a shaker).
2.3.4. Block slides with the blocking solution for 1 hour.
2.3.5. Dilute primary antibody in the blocking buffer per recommendation on the data sheet.
2.3.6. Apply primary antibody to each section and incubate overnight in the humidified chamber (4 ºC).
2.3.7. Wash slides three times with TBST (3 min each on a shaker).
2.3.8. Apply to each section secondary HRP-conjugated anti-rabbit antibody diluted in the blocking solution per
manufacturer’s recommendation; incubate for 1 hour at room temperature.
2.3.9. Wash slides three times with TBST (3 min each on a shaker).
2.3.10. Add freshly prepared DAB substrate to the sections.
2.3.11. Incubate tissue sections with the substrate at room temperature until suitable staining develops (generally 2–5
min).
2.3.12. Rinse sections with water.
2.3.13. Counterstain with Hematoxylin.
2.3.14. Rinse sections with water.
2.3.15. Dehydrate samples using two rinses with 100% Ethanol (20 dips per rinse) followed by two rinses with Xylene (30
dips per rinse).
2.3.16. Mount coverslips on slides using Permount medium.

"We only saw a faint band at 50 kDa using mouse and rat heart, which looks non-specific on our blots. I have not found the identity of this band in the literature. The blocking reagent used was 5% milk. We used our standard WB protocol (see attachment)."

Let me know if you have anymore questions.

Thank you for your reply.
If you were able to detect a different ER protein using the same samples then it doesn't seem like it was a problem with the sample preparation.
I would like to offer you a free of charge replacement of ab108613 (although we only have the same lot in stock as the one you tried), a different GRP78 BiP antibody, or a credit note/refund. Please let me know which you would prefer.
I look forward to your reply.

Thank you for taking time to send us your protocol. I am sorry to hear that this antibody is not providing satisfactory results. The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.
Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab108613.
We would recommend changing your lysis buffer to RIPA buffer which includes SDS. Since this is an ER protein, you're going to need a pretty strong lysis buffer to disrupt the structure and make the protein accessible to the antibody.
RIPA buffer (Radio Immuno Precipitation Assay buffer)
150 mM sodium chloride
1.0% NP-40 or Triton X-100
0.5% sodium deoxycholate
0.1% SDS (sodium dodecyl sulphate)
50 mM Tris, pH 8.0
Also, you do an extra blocking step after the primary antibody incubation but this may not be necessary. Here is the protocol used with this antibody:
Western Blotting

1. Block nitrocellulose for 1 hour at room temperature or overnight at 4°C using 5% BSA.

2.Incubate nitrocellulose with appropriate dilutions of primary antibody in 5% BSA overnight at 4°C or for 2 hours at room temperature
3. Wash nitrocellulose with three 5-min washes using TBST.

4.Incubate nitrocellulose with goat anti-rabbit HRP antibody, diluted to 1:500 to 100,000 in 5% BSA or 5% NFDM, for 1 hour at room temperature.

5. Wash nitrocellulose in 3 washes of TBST, then rinse in TBS prior to addition of chemiluminesence reagents.

6. Remove excess chemiluminescence reagent and sandwich nitrocellulose blot in any type of transparent plastic wrap (plastic cling wrap, transparent sheet protector, etc.).

7. Acquire image using darkroom development techniques.
Should the suggestions not improve the results, please do let me know.
In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.
I hope this information is helpful, and I thank you for your cooperation.

Merci de votre appel. Je suis ravie que vous souhaitiez participer a notre offre et tester ab108613 en immunofluorescence sur des cellules (ICC/IF). Le code de réduction correspondant est xx. Ce code de réduction est utilisable sous 120 jours et pour l’achat d’un autre anticorps primaire.

Comme convenu au téléphone, nous avons aussi validé la commande pour le ab108613.

Vous devez dans un premier temps soumettre une Abreview avec vos résultats en ICC/IF en notant ce code dans la section « Additional notes ». Ce code sera ainsi activé et vous pourrez par la suite contacter notre service clientèle par téléphone pour passer votre prochaine commande, en vous munissant des informations suivantes : code de réduction, numéro de commande de ab108613.
Veuillez consulter le site www.abcam.com/abreviews pour plus d'information sur les abreviews.

Nous restons à votre disposition pour plus d’informations concernant cette offre promotionnelle. Dans l’attente de recevoir votre Abreview nous vous souhaitons bonne chance dans vos recherches.

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Thank you for contacting us, please find the requested CoC attached. Please let me know if you need any other information!