Anti-MCP1 antibody (ab7202)

Thanks for your kindly reply, the information of this product as follow:

Lot: gr47175-3

Po: 1051535

And after I contacted with this customer, she would like to have a credit note, therefore, could you please help her to create a credit note?


I deeply appreciate your great assistance : )

Best regards

ANSWER:

Thank you for your note.



This is to let you know that I have just contacted and asked our Account Department to raise a credit note for you - for the cost of one vial of ab7202. For your information, the internal reference note for this credit is CN20561. You can use this credit in the near future for any of the products which are in the catalogue.



I hope this helps and if I can assist further, please do not hesitate to contact me.

Dear abcam technical support team:

This customer has purchase ab7202 (Anti-MCP1 antibody) and has conducted the WB several times with mouse sample.

The results show no band; moreover, this customer also has used RAW 264.7 cells and stimulated with LPS as a positive control, but the results also shows no band,

she thinks our antibody was lost its function already. Therefore, she would like to ask for your help to solve her problem,

I also attached her data and experiment step with different antibody in this letter, please check this and could you please offer any help to this customer,

thanks for your assistance. Best regards



1. Order details:


Batch number: Lot: GR47175-3

Abcam product code: ab7202

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




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: mouse

What’s cell line or tissue: renal tissue

Cell extract or Nuclear extract: cell extract

Purified protein or Recombinant protein: purified protein




3. The lysate


How much protein was loaded: 150 μg

What lysis buffer was used: RIPA



What protease inhibitors were used: protease inhibitor cocktail, PMSF, NaF NaVO3

What loading buffer was used: SDS sample buffer with protein dye

Phosphatase inhibitors: none

Did you heat the samples: temperature and time: 100℃, 5 min




4. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel: reducing gel

Reducing agent: 2-mercaptoethanol

Gel percentage : 12%

Transfer conditions: (Type of membrane, Protein transfer verified): PVDF membrane, protein transfer apparatus from ATTO corporation (Model: AE6675)




5. Blocking conditions


Buffer: PBS with 0.1 % Tween 20

Blocking agent: milk, BSA, serum, what percentage: 3% BSA

Incubation time: 60 min

Incubation temperature: 25 ℃




6. Primary Antibody


Species: rabbit

Reacts against: MCP-1



At what dilution(s) have you tested this antibody: 1:1000

What dilution buffer was used: 3% BSA in PBS with 0.1% of Tween 20

Incubation time: 18 hours

Incubation temperature: 4 ℃

What washing steps were done: washing membrane with PBS with 0.1% of Tween 20 for 6 times, 5 minutes per each time




7. Secondary Antibody


Species: goat

Reacts against: rabbit antibody

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

Incubation time: 1 hour

Wash steps: washing membrane with PBS with 0.1% of Tween 20 for 6 times, 5 minutes per each time

Fluorochrome or enzyme conjugate: HRP conjugate

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




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



9. Did you apply positive and negative controls along with the samples? Please specify. Yes, the cell lysate of RAW264.7

10. Optimization attempts


How many times have you tried the Western? 2 times

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

Do you obtain the same results every time e.g. are background bands always in the same place? NO

What steps have you altered? NO

ANSWER:

Thank you for your enquiry regarding ab7202 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 antibody.

The protocol looks fine to me. It may well be the antibody has been accidentally damaged.

I can offer a new vial free of charge for your troubled customer but all our vials are from the same belled and purification. Could you please confirm the Abcam Order and the date of purchase and let me know how your customer would like to proceed with this enquiry.

I look forward to hearing from you soon.

No signal in ELISA with ab20641. No signal in WB with ab7202.

ANSWER:

Thank you for your phone call today, and I apologize for any miscommunication with my colleague yesterday. Your credit note ID for ab20641 is: ***. Your credit note ID for ab7202 is: ***. I am sorry that these antibodies did not perform as stated on the datasheet. If payment has already been made on the original orders and you wish to receive a refund, please ask your purchasing department to contact our accounting department so that we may gather the correct information needed for the refund. To avoid confusion, please ensure your accounts department is aware of how the credit note is being used. Our accounting department can be contacted by email at us.credits@abcam.com or by telephone using the information at the Contact Us link in the top right corner of our website. Please refer to the credit note ID in any correspondence with our accounting department. I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service, should you require further expert advice.  

Nuclear staining in mouse spleen and lymph node.

ANSWER:

Thank you for your call today and for your questions about ab7202.

As we discussed, we recommend using a microwave antigen retrieval method with this antibody. However, if the antibody is still staining the wrong localization, please let me know and we will be happy to send a replacement or issue a credit or refund.

I look forward to hearing from you. Let me know if you have any questions.

BATCH NUMBER 7052 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Western blot: no bands detectable Immunohistochemistry: no reaction

SAMPLE WB: mouse aorta protein extract IHC: mouse frozen cross sections of the aorta, OCT embedded

PRIMARY ANTIBODY WB: 1:2000, 1:1000, 1:500 over night, wash step 2x 5min in TBST 0.05% IH: over night 1:400, 1:200, 1:100, wash step 3x 5min in PBS

SECONDARY ANTIBODY WB: Amersham donkey anti-rabbit IgG HRP, 1:3500, 60mion, wash step 1x 15min in TBST 0.05% IH: Vector/Linaris goat anti-rabbit IgG Biotin in PBS, 1:200, 45min or 60min, wash step 3x 5min PBS

DETECTION METHOD WB: ECL IH: DAB-chromogen (Dako)

POSITIVE AND NEGATIVE CONTROLS USED IH: positive: serial sections that showed positive staining with thsi primary asntibody, but different Lot (56165) negative: unspecific IgG

SAMPLE PREPARATION WB: RIPA buffer + protease inhibitor (complete, mini EDTA-free, Roche) 10:1 loading buffer RotiLoad 1 (Roth) 1:4, 95?C, 5min

TRANSFER AND BLOCKING CONDITIONS WB: transfer: transfer buffer (25mM Tris base, 0.2M glycine, 20% methanol, pH 8.5, 60min 340mA or transfer over night 90mA block: 5% milk/TBST, 1h IH: block: 6% goat serum in 4% BSA/PBS or 4% goat serum

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4both HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? WB: first antibody concentration, blocking concentration as indicated IH: first antibody concentration, incubation period, blocking agents as indicated, changed other reagents and tested them iwth another primary antibody in order to find out whether they are still working (positive results).

ANSWER:

I'm sorry to hear the customer is having problems with his new vial of ab7202. Thank you for taking the time to fill in the questionnaire, I can see clearly that the protocol looks very good and the antibody should perform if the customer has previously had good results.

Could you please confirm the lot number or the order number for this vial as we have no record of a lot 7052 and confirm the storage conditions, my apologies for the inconvenience. Could you please also send us an image of the positive staining with lot 56165?

Thank you for your patience, we look forward to hearing from you soon,