Anti-pan-AKT (phospho T308) antibody (ab38449)

Our customer is using this antibody in WB and detecting no bands. They have made the following modifications based on your suggestions:

Block and dilute primary and secondary antibodies in 5% BSA

ANSWER:

Thank you for your reply.

I have reviewed the customer's comments and image and agree that this antibody is not working as stated on the datasheet.  I am happy to offer a replacement or credit.  Please let me know which the customer would prefer.

I look forward to your reply so that I may assist you further.

Dear Tech Support Team,   Our customer is willing to test ab38449 in ICC/IF with mouse cells (MIN6, islets). Are you interested in this type of the information for the Abreview promotion (The customer buys certain antibodies and upload Abreview on Abcam's website and then we give 100% credit note for this antibody to buy next antibody)? Thanks in advance for your assistance and reply. Best Regards,

ANSWER:

Thank you for contacting Abcam. We would be very happy if your customer tested ab38449 in ICC/IF in mouse cells. I have placed the discount code and instructions below. If there is anything else I can help you with please let me know. DISCOUNT CODE: **** Expiration date: ******I am very pleased to hear you would like to accept our offer and test ab38449 in ICC/IF in mouse cells. This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for ICC/IF in mouse cells and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews. Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

 Dear tech,   A customer had problems from ab38449.  

1)     Abcam product code  ab 38449

3) Storage conditions: -20℃ 4) Description of the problem (high background, no band, wrong band size...) No bands     5) Sample preparation: Name of the tissue/cell line:B16 Sample species:mouse Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysates Lysis buffer :RIPA Buffer Protease inhibitors:PMSF Phosphatase inhibitors :NaF Reducing agent: bromophenol blue             

Boiling for ≥5 min? yes  Protein loaded ug/lane or cells/lane: 50 ug Positive control

Negative control

  6) Percentage of gel:10% Type of membrane PVDF Protein transfer verified       Wet,250mA,90min

Blocking agent and concentration: 5% nonfat dried milk（TBST dilution） Blocking time  1h                     Blocking temperature room tempreture   7) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution:1:500

 Diluent buffer  5%BSA(TBST dilution) Incubation time: overnight Incubation temperature:4℃   8)  Secondary antibody:     

Species: rabbit Reacts against: goat Concentration or dilution:  1:5000 Diluent buffer  5%BSA(TBST dilution) Incubation time: 1h Incubation temperature: room tempreture Fluorochrome or enzyme conjugate: Horseradish Peroxidase (HRP)   9) Washing after primary and secondary antibodies: Buffer: TBST Number of washes: 10min×3   10)Detection method ECL 11) How many times have you run this staining? Three times. Do you obtain the same results every time? yes

What steps have you altered to try and optimize the use of this antibody?

Increase the antibody concentration and reduce washing times.

ANSWER:

Thank you for contacting Abcam.

I am sorry that your customer is experiencing difficulties with this antibody in WB. I have reviewed the protocol information provided and would like to make some suggestions to improve the results with this product.

Regarding the customer's sample - is this considered a positive control? Do you expect AKT to be phosphorylated? Is the customer treating the samples in any way to induce phosphorylation? Has a positive and negative control been run? Also, the customer has indicated that bromophenol blue is used as a reducing agent, however this reagent is not a reducing agent. Is any beta-mercaptoethanol or DTT included in the sample buffer?

Since the customer is using a phosphospecific antibody, it is not suggested to use milk as a blocking reagent. Although BSA has been used as the antibody diluent, I would recommend using BSA as a blocking reagent as well.

Finally, the customer indicates that they are using a rabbit anti-goat secondary antibody. Since the primary antibody is raised in rabbit, the customer must use an anti-rabbit secondary antibody.

I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions or if these suggestions do not improve the results.

I am interested in AKT (phospho T308) antibody - ab38449. I am planning to use it on Entamoeba histolytica and I would like to check if it's going to cross react. Could you please send me a recognition protein sequence for this antibody? Thank you for your help,

ANSWER:

Thank you for your enquiry.

I have contacted the originator of the product but unfortunately, the exact peptide sequence information is proprietary. However, based on the sequence we found for Entamoeba histolytica (swiss prot#Q761W9: Putative uncharacterized protein / Protein kinase AKT), seven amino acids in a row around the phosphorylation site T 308 (TFCGTPE) are the same with the designed peptide, which is around 54% homology.

I hope this information will still be useful to you.

If there is anything else that I can help you with, please do not hesitate to contact me.