Rabbit Anti-Mouse IgG H&L (HRP) (ab6728)

Thank you for your email. No, ab6728 is not pre-adsorbed. If you have any additional questions, please contact us again.

Thank you for your email. Ab11026 - Mouse monoclonal antibody to Aquaporin 4 - is currently in stock and is $299 for 50 ug. Ab6728 - Rabbit polyclonal antibody to Mouse IgG H&L (HRP) - is also currently in stock and is $150 for 1 mg. Ab11026 has been tested for application in Western blotting and found to cross-react with human. If you go ahead and try this antibody with your samples, I do suggest running a positive control; kidney tissue is recommended. Ab6728 is a suitable secondary antibody to use with this primary. If you have any additional questions, please contact us again.

Thank you for your email. At this point I would like to offer you a replacement vial free of charge or a refund, please let me know which you would prefer. Thanks again, and I look forward to hearing from you.

Thank you for your email. I'm replying to with regard to this issue as my colleague Tanya is away for the Easter holidays. With the HUVEC lysate, what dilutions of ab10966 did you try? Did you incubate overnight at 4C? I apologize if this information was in your previous email, the bottom may have been accidently deleted. Thanks again, and have a great weekend.

Thank you for contacting us and for providing details of your Western blot protocol. We are very sorry to hear that you are having problem with this antibody. We have read through your message and would like to make the following suggestions/comments: 1. Although we suggest using this antibody at a concentration of 1 - 2 µg/ml, this range is just a recommendation. This product was tested and characterised using recombinant protein. The detection limit for recombinant human placental growth factor is approximately 20 ng/lane and 300 ng/lane under non-reducing and reducing conditions, respectively. We would strongly suggest running purified recombinant protein as positive control along with your samples. 2. If this target protein is expressed at lower levels, then you need to optimize the concentration. Do you know the expressed levels of the Placental Growth Factor in the keratinocytes? If you can't have recombinant protein, then you need to use Human umbilical vein endothelial cells as positive control. 3. You have not mentioned in your e-mail from which species the cells come from? Are they human, mouse, rat etc? This antibody recognizes only human Placental Growth Factor, we have not tested the cross-reactivity with other species. 4. Always use freshly made lysis buffer with a cocktail of proteinase inhibitors. We normally suggest using RIPA buffer. 5. Incubate the membrane with the primary antibody overnight at 4oC to see if the signal is getting stronger or not. 6. Have you tested the transfer of the proteins with Ponceau or other type of dye? How do the molecular weight markers look like? 7. Please also test the secondary antibody with another primary antibody. 8. Finally, we would suggest testing whole cell lysate (make samples before the centrifugation step in case the target you may lose some material in the pellet). Load 20-30 ug per ml of total protein per lane but not more than that other wise the gel may be overloaded. We hope this information will be useful for you. Please find enclose Preparation of RIPA cell lysates for Western Blots: 1. Wash cell pellets once with ice-cold PBS. 2. Add 1 ml of RIPA buffer to 10x8 cells, incubate on ice for 20 min, vortex 2 to 3 times. 3. Centrifuge for 5 min at 4oC at maximum speed in a microfuge tube. 4. Transfer supernatant into clean tube. Measure protein concentration with a protein assay. 5. Adjust concentration to 5 mg/ml with RIPA lysis buffer. 6. Add equal volume of 2 x SDS sample buffer into cell lysate, boil for 5 min. 7. Store at -20oC for daily use or -80oC for long term. Avoid repeated freeze thaw cycles. RIPA Base Ingredients Tris-HCl: 50 mM, pH 7.4 NP-40: 1% Na-deoxycholate: 0.25% NaCl: 150 mM EDTA: 1 mM PMSF: 1 mM Aprotinin, leupeptin, pepstatin: 1 microgram/ml each RIPA Protease Inhibitors Phenylmethylsulfonyl fluoride (PMSF) (200 mM stock solution in isopropanol; store at room temperature) EDTA (calcium chelator; 100 mM stock solution in H2O, pH 7.4) Leupeptin (store frozen in aliquots, 1 mg/ml in H2O) Aprotinin (store frozen in aliquots, 1 mg/ml in H2O) Pepstatin (store frozen in aliquots, 1 mg/ml in methanol) Good luck! Should you need further assistance, then please do not hesitate to contact us again.

Thank you for your email. You don't have to conjugate the primary antibody, you can do an indirect ELISA where you use an alkaline phosphatase-conjugated secondary antibody or peroxidase-conjugated secondary antibody. Below is our protocol for indirect ELISA. Please contact us again if you have any additional questions. ELISA 1. Dilute the antigen to a final concentration of 20 µg/ml in PBS. Coat the wells of a PVC microtiter plate with the antigen by pipeting 50 µl of the antigen dilution per well. 2. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature. 3. Remove the coating solution and wash the plate twice by filling the wells with 300 µl PBS. The solutions or washes are removed by flicking the plate over a sink. The remaining drops are removed by patting the plate on a paper towel. 4. Block the remaining protein-binding sites in the coated wells by adding 300 µl blocking buffer, 5% non fat dry milk/PBS, per well. 5. Cover the plate with an adhesive plastic and incubate for at least 2 h at room temperature or, if more convenient, overnight at 4°C. 6. Wash the plate twice with PBS. 7. Make 10-fold dilutions (1:100, 1:1,000, 1:10,000, 1:100,000 and 1:1,000,000) of serum and pre-immune serum (the latter as a negative control) in blocking buffer. Add 50 µl of each dilution to an antigen-coated well. 8. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature. 9. Wash the plate four times with PBS. 10. Add 50 µl of secondary antispecies antibody conjugated to alkaline phosphatase, diluted at the optimal concentration (according to the manufacturer) in blocking buffer immediately before use. 11. Cover the plate with an adhesive plastic and incubate for 2 h at room temperature. 12. Wash the plate four times with PBS. 13. Dissolve p-Nitrophenyl phosphate at a concentration of 1 mg/ml in substrate buffer (1M diethanolamine, 0.5 mM MgCl2, pH 9.8). Add 50 µl of the substrate solution per well with a multichannel pipet or a multipipet. 14. Measure the absorbance at 405 nm, using a microtiter plate spectrophotometer. Perform an end-point measurement after 1 h. 15. Calculate the titer of the sera. The titer can be defined as the dilution of serum giving an optical density (OD) of 0.2 above the background of the ELISA after a 1-h reaction.

Thank you for your enquiry. It depends on what the primary antibody is - would you be using a mouse monoclonal? If you are, this is a good one to try. Just so you know, at the bottom of the datasheets for all our primary antibodies there is a list of compatible secondaries which can be used.

Thank you for your email. We have received feedback from customers who have used this antibody that dilutions from 1:2000 to 1:5000 have worked very well for them. You may read these reviews by clicking on the reviews tab located on the online datasheet for ab6728. I would therefore suggest starting with a dilution of 1:3000 and optimizing from there. If you have any additional questions, please contact us again.

The concentration is 2.00 mg/ml. There's a review on the datasheet for this product where another researcher recommends a 1:3000 dilution. However, we always recommend that conditions for WB should be optimised by the user.