Anti-XRCC2 antibody [XRCC2 1G4/1] (ab20253)

Dear technical support team:

This customer has purchased ab20253 (Anti-XRCC2 antibody [XRCC2 1G4/1])and has conducted the WB several times with human sample. The results show high background and more bands; therefore this customer wants to ask for your help to modify her experiment step, could you please offer any suggestion to improve her result?

I attached the image in this letter and her experiment step as follow:

1. Order details:


Batch number: xxxx

po: xxxx

Abcam product code: ab20253

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




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

High background, more bands, NO bands



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

· Species: human

· What’s cell line or tissue: HepG2 cell line

· Cell extract or Nuclear extract: cell extract

· Purified protein or Recombinant protein: purified protein



3. The lysate


How much protein was loaded: 40ug

What lysis buffer was used: sigma cellytic mt cell lysis reagent



What protease inhibitors were used: sigma protease inhibitor cocktall without*metal chelating

What loading buffer was used: no

Phosphatase inhibitors: no

Did you heat the samples: temperature and time: 95℃, 5 minutes




4. Electrophoresis/Gel conditions/ Transfer conditions


Reducing or non reducing gel: non reducing gel

Reducing agent:

Gel percentage : 10% gel

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


PVDF membrane



5. Blocking conditions


Buffer: PBST

Blocking agent: milk, BSA, serum, what percentage: VISUAL protein blotechnology CORP BlockPRO blocking buffer

Incubation time: 2hrs

Incubation temperature: RT for 25℃




6. Primary Antibody


Species: human

Reacts against: anti-mouse


· At what dilution(s) have you tested this antibody: anti-XRCC2:PBST=1:200

· What dilution buffer was used: PBST

· Incubation time: overnight

· Incubation temperature: 4℃

· What washing steps were done: PBST wash 3 times/10 minutes



7. Secondary Antibody


Species: human

Reacts against: HRP-ECL

At what dilution(s) have you tested this antibody: anti-mouse:PBST=1:5000 Incubation time: 1hrs

Wash steps: PBST wash 3 times/10 minutes

Fluorochrome or enzyme conjugate: HRP-ECL

Do you know whether the problems you are experiencing come from the secondary? I do not know




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

ECL

9. Did you apply positive and negative controls along with the samples? Please specify. no

10. Optimization attempts

· How many times have you tried the Western? 4-5 times

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

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

· What steps have you altered? Primary antibody 1:500 and 1:200



Could you please help this customer to solve the problem?

Thanks for your kindly help.

Best regards.

ANSWER:

Thank you and your customer for taking the time to complete our questionnaire and contact us. I am sorry to hear your customer has had difficulty obtaining satisfactory results from this antibody.

The details your customer has kindly provided will enable us to investigate this case for your customer and this is also helpful in our records for monitoring of quality.

Reviewing this case, I would like to offer some suggestions to help optimize the results from ab20253.

Reviewing the image I believe it was very overexposed as judged by the appearance of the standard as one solid black line. In my opinion the actual problem is not multiple bands but no band.

XRCC2 is a nuclearprotein and requires harsh detergents to be isolated from the nucleus. I recommend a RIPA buffer with SDS.

The lysis buffer used is described as: "Gentle: Non-denaturing and does not interfere with downstream applications" and "The lysis buffer consists of a dialyzable mild detergent,..." by the company that sells it.

I think it is not suitable for nuclear proteins.

I can recommend that your customer prepares nuclear fractions instead.

We are happy to offer this technical support. 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 and your customer for your cooperation. Should the suggestions not improve the results, please do not hesitate to contact me again.I will be happy to provide more protocol tips.