Anti-AHRR antibody (ab85666)

Thanks for your kindly reply, after I contacted with this customer, she replied your question as follow:   1. Storage at -20 2. Shipped at 4°C and store at -20°C 3. I think this detection system is fine.   This customer is already tried this experiment over then three times, and the results show weak signal even she increased the loading protein to 50ug, could you please help this customer to solve the problem? Thanks for your kindly help Best regards

ANSWER:

Thank you for getting back to me and for passing some further information.

I could offer either a new vial free of charge or a credit note which can be used for future purchase. Please could you get back to your troubled customer and discuss how he/she would like to proceed with this case.

I look forward to hearing from you so

LOT NUMBER gr54067-1

ORDER NUMBER 937542

DESCRIPTION OF THE PROBLEM

No signal or weak signal and no band in positive control (HepG2 cell lysate)

SAMPLE

•Species: human

•What’s cell line or tissue SK-Hep1

•Cell extract or Nuclear extract: total protein cell lysate

•Purified protein or Recombinant protein: no

PRIMARY ANTIBODY

•Species: Rabbit

•Reacts against:

•At what dilution(s) have you tested this antibody: 1 ug/ml

•What dilution buffer was used: 5% no fat milk in TBS

•Incubation time: overnight

•Incubation temperature: 4℃

•What washing steps were done: 5 min/time with TBST (0.1% tritonX20), 5 times

DETECTION METHOD

ECL+

POSITIVE AND NEGATIVE CONTROLS USED

Yes. (50 μg HepG2 cell lysate)

ANTIBODY STORAGE CONDITIONS

937542

SAMPLE PREPARATION

•What lysis buffer was used: RIPA buffer

•What protease inhibitors were used: Aprotenin, Leupeptin, PMSF

•What loading buffer was used: β-ME

•Phosphatase inhibitors: β-glycerophosphate, Na3VO4

•Did you heat the samples: temperature and time: 95℃ 5min

AMOUNT OF PROTEIN LOADED

50 μg

ELECTROPHORESIS/GEL CONDITIONS

•Reducing or non reducing gel: non reducing gel

•Reducing agent:

•Gel percentage : 10% SDS-PAGE

TRANSFER AND BLOCKING CONDITIONS

•Transfer conditions: (Type of membrane, Protein transfer verified): PVDF, transfer condition: 4℃ 300 mA 2hr

•Buffer: TBS

•Blocking agent: milk, BSA, serum, what percentage: 5% no fat milk

•Incubation time: 1 hr

•Incubation temperature: RT

SECONDARY ANTIBODY

•Species:goat

•Reacts against: rabbit

•At what dilution(s) have you tested this antibody: 1:3000

•Incubation time: 2 hr

•Wash steps: 5 min/time with TBST (0.1% tritonX20), 5 times

•Fluorochrome or enzyme conjugate: enzyme conjugate

•Do you know whether the problems you are experiencing come from the secondary? No

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?

protein loading

ANSWER:

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is having problems with this product.

Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.

ANTIBODY STORAGE CONDITIONS

937542

Questions:

- Could you please clarify how this product was stored and shipped to the end-user?

- Does the detection system work fine? Have you used it successfully with another primary antibody?

I look forward to hearing from you and hope to solve this problem as soon as possible.