Anti-XRCC1 antibody (ab9147)

> Could we get a detailed protocol from you, please?

Harvest Cells:

wash cells with pbs, add RIPA Buffer, refrigerate for 10-15 minutes until cell appear lysed, homogenize sample with 201/2-gauge needle, spin down and take off supernatant, quantitate protein concentration with the lowry method typically get 5-7 mg/ml from a 35 mm plate

gel:

load 30 micrograms of protein, 100v until through stack, 250v in resolving gel, semi-dry transfer to pvdf

blotting: block for 1 hour in pbst + 5% milk XRCC1 polyclonal 1:5000 (ab9147-50) in pbst + 5% milk for 1.5 hours wash with pbst for 5 min, repeat 5 X Santa Cruz anti rabbit IgG hrp conjugate (sc2030) 1:3000 for 1.5 hours wash with pbst for 5 min, repeat X 5 expose to substrate for 1 min, put on film for 5 min

i've used this protocol for about two years now on both helas and 293's.

Also, please include the previous lot numbers that you used (if > you still have the information) and the Abcam order number or > purchase order number that was used for your latest order for > ab9147.

unfortunately i don't have the previous lot numbers as i only use about 1 vial per year (i've found that i can re-use the primary 2-3 time with good results). The order reference number for the most recent lot is 19207 and the abcam order number is 53805.

> > > > 1. Please describe the problem (high background, wrong band size, > more bands, no band etc).

No signal at all, no background, nothing

> 2. On what material are you testing the antibody in WB?

cell extract from 293's

> 3. How much protein did you load? 30 micrograms

> â?¢How did you prepare the lysate for the analysis (protease > inhibitors etc)?

wash cells with pbs, ripa buffer with PMSF, refrig. 10-15 min, homogenize with 201/2 gauge needle, spin down, take supernatant, quantitate protein

> â?¢Did you heat the samples?

yes

> 4. Primary Antibody > â?¢Specification (in which species was it raised against)? human > â?¢At what dilution(s) have you tested this antibody? i normally use at 1:5000 > â?¢Incubation time, wash step? 1.5 hours, yes i wash 5 times, for 5 minn with pbst > > 5. Secondary Antibody > â?¢Specification (in which species was it raised against)? Santa Crus Anti-rabbit IgG-hrp SC2030 > â?¢At what dilution(s) have you tested this antibody? 1:3000 > â?¢Incubation time, wash step? 1.5 hr, yes, as above > â?¢Do you know whether the problems you are experiencing come > from the secondary? i've recently used the secondary with other polyclonal primaries with no problems (less than a week) > > 6. What detection method are you using? >ECL Western Blotting Analysis Kit from Amersham RPN2108 > 7. Background bands >NA â?¢Please provide an image of your blot (as an e-mail attachment, > a faxed image is not sufficient) see attached. > > 8. Optimization attempts > â?¢How many times have you tried the Western? I've been using this antibody successfully for two years, the most recent blots with the new antibody are my only failures.

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

yes, i have positive controls on every blot (both nuclear extract from hela cells and whole cell extract from control 293 cells)

Thanks

ANSWER:

Thank you for the details in which you have provided. Since you got absolutely no signal even with the HeLa nuclear extract (what is recommended as a positive control) it's definitely telling me that something is wrong with the batch/vial that you received. My suggestion would be to try increasing the concentration of the primary antibody, but since you're not even seeing any background I really don't think that would help. Anyway, I'm sending a replacement vial to you on order# 54735 and you should receive it tomorrow. Please let me know how it works out for you and if you have any further questions.