Anti-E Cadherin antibody (ab15148)

Can you please tell me which of your human E Cadherin antibodies recognize the extracellular domain?

ANSWER:

Thank you for your phone call. Our current E Cadherin antibodies that bind to an extracellular domain of the human protein are:

ab8050
ab14015
ab15148
ab15150
ab22585
ab40772
ab45990
ab77287

I hope this helps, please let me know if you would like me to narrow down the list by any additional criteria.

Thanks for your kindly reply, after I contacted with this customer, she would like to have a credit note. Therefore, would you please help this customer to create a credit note? Thanks for your kindly assistance.  

ANSWER:

Thank you for confirming this information and for your help and cooperation with this case.

As requested, I have asked our accounting department to issue you with a credit note. This can then be redeemed against the invoice of a future order.

As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. The reference number, or credit ID, is xxxx. Please refer to this number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.

Thanks for your kindly reply, after I contacted with this customer, she reply your question as follow: 1.    Positive control: Huh7, ab1416:the user says it can work 2.    Yes, we have checked E-cadherin expressed in Huh7 by real time PCR. Furthermore, this customer indicated she also used the ab53033 and conducted wb assay with the same condition as this time, and the results show strong signal, I attached the image in this letter, could you please help this customer to solve the problem?  

ANSWER:

Thank you for confirming this information and for your help and cooperation with this case.

As mentioned before, the protocol looks absolutely fine to me and the results with ab53033 are excellent. I can suggest the customer has regrettably received a bad vial. Therefore, I would be pleased to offer your customer a replacement or a credit note.

I look forward to your reply.  

LOT NUMBER gr46326-1 ORDER NUMBER xxxxDESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE •Species: human cell line •What’s cell line or tissue: human HCC cell line ex:huh7 hucct1 •Cell extract or Nuclear extract: cell extract PRIMARY ANTIBODY •Reacts against:anti-E cadherin •At what dilution(s) have you tested this antibody:1:500 •What dilution buffer was used: 5% skim milk in PBST •Incubation time: overnight >16hr •Incubation temperature: 4℃ •What washing steps were done:4 times, 10min every time DETECTION METHOD ECL ANTIBODY STORAGE CONDITIONS -20℃ SAMPLE PREPARATION •What lysis buffer was used: TPER+phospho stop+EDTA free •What protease inhibitors were used: phospho stop+EDTA free •What loading buffer was used: •Phosphatase inhibitors •Did you heat the samples: temperature and time: 95℃ 5min AMOUNT OF PROTEIN LOADED 66μg ELECTROPHORESIS/GEL CONDITIONS •Gel percentage : 6 TRANSFER AND BLOCKING CONDITIONS •Transfer conditions:PVDF, 100volt 90min •Buffer:5% skim milk in PBST •Blocking agent: milk, BSA, serum, what percentage: 5% skim milk •Incubation time:1hr •Incubation temperature:room temperature SECONDARY ANTIBODY •Reacts against:anti-rabbit IgG •At what dilution(s) have you tested this antibody: 1:10000 •Incubation time: 1hr •Wash steps: 4 times, 10min every time •Fluorochrome or enzyme conjugate:HRP HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Dilution (from 1:1000 to 1:500), but results are still the same result

ANSWER:

Thank you for contacting us.

I am sorry to hear you have been experiencing problems with one of our antibodies. The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints.

I appreciate the time taken to complete our questionnaire, and after reading the answers provided I would like to ask additional questions:

1) Could you please confirm which positive control you have used?

2) May I ask whether you have checked your cell lines for the transcription of E-Cadherin, e.g. by RT-PCR?

As your protocol looks absolutely fine to me, I would be pleased to offer you a replacement or a credit note, once I have received your reply.

I look forward to hearing from you again.

Hi,   I am attaching the questionnaire in this mail. Looking forward to hear from you.   Best,  

1) Abcam product code  ab Rabbit polyclonal to E Cadherin (Ab15148)                     

2) Abcam order reference number or product batch number Lot# GR47789-1  

3) Description of the problem: no band / high background (many non-specific bands) / wrong band size / other? Wrong band size.  

4) Sample preparation: Type of sample (whole cell lysates, fraction, recombinant protein…): Species : Human (MCF7 cells) Lysis buffer : RIPA (Cell Signalling, #9806)

Protease inhibitors:  Sodium deoxycholate, leupeptin (already included in RIPA buffer) Phosphatase inhibitors : PMSF (sodium pyrophosphate, beta-glycerophosphate & Na3VO4 are aready included in RIPA buffer) Reducing agent : DTT                      Boiling for ≥5 min? yes/no  

Protein loaded ug/lane or cells/lane :40ug/lane               

Positive control :No Negative control : No.    

5) Percentage of gel 4%-12% NuPAGE gel Type of membrane Nitrocellulose Membrane

Protein transfer verified          Yes. With Ponceau Stain              Blocking agent and concentration 5% Non-Fat milk in 1xPBS Blocking time 15min                                        Blocking temperature room temperature    

6) Primary antibody (If more than one was used, describe in “additional notes”) : Concentration or dilution 1:500  Diluent buffer  5% non-fat milk in 1xPBS Incubation time overnight Incubation temperature:4C    

7)  Secondary antibody:                Species:Goat Reacts against: rabbit Concentration or dilution 1:2000

Diluent buffer  5% non-fat milk in 1xPBS Incubation time 1.5h Incubation temperature: room temperature Fluorochrome or enzyme conjugate: HRP    

8) Washing after primary and secondary antibodies: Buffer 5% non-fat milk in 1xPBS Number of washes 3 times each, 5 minutes per time         

9)Detection method SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Prod# 34080)  

10) How many times have you run this staining? twice

Do you obtain the same results every time?  Yes

What steps have you altered to try and optimize the use of this antibody? Transfer time, optimized from 1h to 1.5h  

ANSWER:

Thank you for kindly providing these details. Your enquiry has been forwarded to me as my colleague is currently away from the office.

Reviewing this case, I would like to offer some suggestions to help optimise the results from ab15148. I would also appreciate if you can confirm some further details:

1. Could you confirm if the washes are in PBS containing 0.1% Tween? I can suggest to also include tween in the antibody dilution buffers. This will help to keep the antibody solubilized and also wash away any excess antibody.

2. To help reduce any non specific staining, I can suggest to try the antibody at a lower concentration of 1:1000.

3. This protein is cleaved by several proteases, which may explain the extra bands and the results that you have. Therefore I can suggest to consider trying again from a fresh sample. Harvest the cells for lysis when they are about 70-80% confluent. Overconfluent cells can contain degraded proteins.

The SwissProt/Uniprot site states the following regarding cleavage:

700 – 701 Cleavage; by a metalloproteinase Fragment sizes: amino acids 1 - 700: 77 kDa amino acids 701-882: 20 kDa

731 – 732 Cleavage; by gamma-secretase/PS1 Fragment sizes: amino acids 1-731: 80 kDa amino acids 731-882: 17 kDa

750 – 751 Cleavage; by caspase-3 Fragment sizes: amino acids 1 -750: 83 kDa Amino acids 750 – 882: 15 kDa

Although these are not the exact band sizes seen on your blot, with the glycosyation expected with this protein, these fragments could appear at a higher molecular weight. There may also be other proteins that cleave E cadherin.

5. Could you confirm if the current vial of secondary antibody and the detection kit are working well with other primary antibodies? I can recommend to include a no primary control to asses if the secondary is binding non specifically. The concentration of secondary may need to be reduced in order to optimize the results.

In the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I hope this information is helpful, thank you for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again with the further requested details.