Anti-HEY1 antibody (ab22614)

Dear technical support:

This customer raised an inquiry about ab22614 (Anti-HEY1 antibody), and this customer used this antibody conduct the western blotassay with human sample, and the data shows no band, because this antibody is already out of our warranty period, therefore she only wants to ask for your kindly help to offer any suggestion to her. I attach the WB questionnaire in this letter, could you please help this customer to solve this problem.

Thanks for your kindly assistance.

Best regards

1. Order details:

Batch number: GR20993-4

Po: 968560

Abcam product code: ab22614

Antibody storage conditions (temperature/reconstitution etc) : -20 degrees

2. Please describe the problem (high background, wrong band size, more bands, no band etc).

No band

3. On what material are you testing the antibody in WB?

· Species: human

· What’s cell line or tissue: cell line

Huh7 / Hep3B / HepG2 / Ha22T / SKHEP1 /HCC26 / Mahlavue

· Cell extract or Nuclear extract: cell extract

· Purified protein or Recombinant protein: purified protein

3. The lysate


How much protein was loaded:

What lysis buffer was used: RIPA lysis buffer

What protease inhibitors were used: PMSF

What loading buffer was used: 5x SDS loading dye

Phosphatase inhibitors : no

Did you heat the samples: temperature and time: 98 degrees / 10min

4. Electrophoresis/Gel conditions/ Transfer conditions

Reducing or non reducing gel: reducing gel

Reducing agent: TEMED

Gel percentage : 10 % PAGE

Transfer conditions: (Type of membrane, Protein transfer verified):

Wet transfer / 110v 90min limit:0.33amp / PVDF transfer membrane

5. Blocking conditions

Buffer: 1X TBST

Blocking agent: milk, BSA, serum, what percentage: 5% milk

Incubation time: 60 min`

Incubation temperature: room temperature

6. Primary Antibody

Species: rabbit

Reacts against:anti-HEY1

· At what dilution(s) have you tested this antibody: 100x/200x/500x

· What dilution buffer was used: TBST

· Incubation time: over night

· Incubation temperature: 4 degrees

· What washing steps were done: TBST wash 10 min / 3 times

7. Secondary Antibody

Species: goat

Reacts against: anti-rabbit IgG HRP

At what dilution(s) have you tested this antibody: 10000x

Incubation time: RT

Wash steps: TBST wash 10min / 3 times

Fluorochrome or enzyme conjugate: HRP conjugated

Do you know whether the problems you are experiencing come from the secondary? I am sure that secondary Ab does work.

8. Detection method
ECl, ECl+, other detection method: ECL

9. Did you apply positive and negative controls along with the samples? Please specify.

Positive control : alpha-tubulin

10. Optimization attempts

· How many times have you tried the Western? 3 times

· Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control):

I am sure that secondary Ab works.

· Do you obtain the same results every time e.g. are background bands always in the same place? Repeated the same results ( No band)

· What steps have you altered? Decrease Ab dilution -> No band

ANSWER:

Thank you for taking time to complete our questionnaire and informing us of the problems that your customer has been having with this antibody.

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 also appreciate it if youcould confirm some further details:

1. You said that both whole cell extract and recombinant protein was used to perform the blot? How was the recombinant protein produced? Is it possible to share an image of the blot obtained, along with that produced with the alpha-tubulin control?

2. How much protein was loaded in each well?

3. Has the transfer been verified by staining of the membrane?

4. Was thealpha-tubulinprobed on the same membrane?If so was a stripping procedure employed? Which protein target was probed first?

5. How long was the membraneexposed for?

Once I have this additional information I will be able to give the customer a few suggestions which may help to improve the results observed so far.

I thank you for your cooperation and look forward to your reply.

P {margin-top:0;margin-bottom:0;} Hello, I bought HEY1 antibody (ab22614) in your company. Would you please recommend good negative control for IHC or IHF staining? Thank you very much Sincerely,    

ANSWER:

Thank you for contacting us. When consulting the images provided on the datasheet of ab22614, it seems to that these tissues (lung and kidney) do have a specific staining pattern - and should therefore provide an internal negative control in my opinion. Indeed, the kidney seems to have only expression in the epithelial cells of the renal tubules and not so much in the underlying cells.  I am however not a specialist in the domain, and I do not want to recommend a control that may be unsuitable. I therefore encourage you to consult the latest literature available through PubMed and other resources in order to find the most up-to-date information about the exact expression pattern of HEY1. Please find here for example the information from Swissprot in regards to tissue expression of HEY1 as well as the link to the original publications:  Tissue specificity Expressed in somitic mesoderm, brain, central nervous system, kidney, heart, nasal epithelium, limbs, lung, muscle, ovary and testis. [PubMed: 10860664] PubMed: 12947105] Developmental stage Expressed in the developing nervous system, the somites, heart and craniofacial region. Expressed in the myocardium of the atrium at E9.5 and in the atrioventricular cushions from E9.5 to E12.5. Also expressed in developing and adult retina. Expressed in the ventricular zone of the ventral spinal cord at E12 and in the ventricular zone of the telencephalon at E12 and E15. Weakly expressed in the cortical plate at E15 Once you have decided about the best tissue to use in your case, I would be happy to search for the corresponding tissue slides in our catalogue. We do have for example lung tissue slides, such as the ab4349. Click here (or use the following: http://www.abcam.com/index.html?datasheet=4349). I am sorry that I could not be more helpful, but I hope that the available literature in this area can provide some clarification. Please do not hesitate to contact us again with other needs or with any questions about our products. 

Would like to use ab98072 in Western blot with rat samples.

ANSWER:

DISCOUNT CODE: *** Expiration date: January 13, 2012

I am pleased you would like to accept our offer and test ab98072 in rat. This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for ab98072 in rat and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. 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.