Anti-RING2 / RING1B / RNF2抗体(ab3832)

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this antibody.

I would like to reassure you that this antibody is tested and covered by our 6 month guarantee forWB and in human and mouse samples. In the event that a product is not functioning in the tested applications and species 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 would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records.with furhter information, I woudl be more able to provide further suggestion onthe western blot. In order to proceed with this, I have enclosed a technical questionnaire below. I would appreciate if you are able to complete this. It will help you put the information we require together very easily.

Thank you for your time and cooperation. We look forward to receiving the completed questionaire.



Order Details
Antibody code:RING1b (ab3832) antibody

Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded
20µg

Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)

1:500 in PBS-Tween for overnight incubation.

Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)

HCL exposure

Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

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


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

Thank you for contacting us.
I am sorry to hear the antibody does not give you results as expected.

I do have a couple questions in order to understand your experiment better:

1) Are you using the antibody for IP or ChIP? There are differences between both techniques including the cross-linking step and the detection method. IP by itself has not been tested with this antibody.

2) Are you using WB (for IP) to obtain your results or rather RT-PCR (for ChIP)?
3) Also, what controls have you included in your experiment, especially when doing ChIP?
4) Could you please send me your protocol?

I look forward to hear back from you.

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Thank you for your enquiry. I have consulted the EXPASY databases and I retrieved the following information: Mel18 - Tissue specificity: Detected in all tissues examined with high expression found in placenta, lung and kidney and low expression, in liver, pancreas and skeletal muscle. Ring1B - No tissue specificity information. The recommended positive control for Mel18 is human lung lysate. For Ring1B we do not have a recommended positive control. However, a reviewer has successfully used this antiserum in western blotting using mouse cultured cells (3T3) and mouse brain. This may be a useful alternative to HeLa cells as I cannot guarantee that these cells express Ring1B. I have obtained the following details of how the positive control to how Mel18 was prepared and run by western blotting. Western Blot: Approx 40 kDa band observed in Human Lung extracts (predicted MW of 40kDa according to NP_009075). Recommended for use at 1-2µg/ml. Tissue Lysis. Tissue chunks were weighed and cut into approx 1mm cubes using a razor blade. The tissue was transferred to a handheld homogenizer and 3 ml of ice-cold RIPA buffer was added per 1g of tissue. The tissue was gently homogenised over 20 minutes on ice. The resulting lysate was aliquotted into 1.5 ml microfuge tubes and centrifuged at 13,000 rpm for 5 min in a microfuge. The supernatant was transferred into clean tubes and its protein concentration was measured with BioRad protein assay. The concentration was then adjusted to 5 mg/ml with RIPA lysis buffer. An equal volume of 2 x SDS sample buffer was added and the cell lysate was boiled for 5 minutes. Lysates were stored at -80C until use.(RIPA buffer = 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 1 mM EDTA, 5 µg/ml Aprotinin, 5 µg/ml Leupeptin, 1% Triton X-100, 1% Sodium deoxycholate, 0.1% SDS). SDS PAGE. Samples were run at 200V constant on a 12% acrylamide SDS-PAGE mini gel - using Biorad Mini-Protean 3 kit and protocols. Before loading samples had 5% (v/v) 2-ME added and were boiled for 3 minutes. Transfer. We used a Biorad Mini Trans-Blot, constant 100 V for 1 hour. Transfer Buffer was 20 mM Tris pH 8.0, 150 mM Glycine, 10% Methanol. We transferred to Millipore PVDF membrane and stained with Ponceau Red to evaluate the transfer. Staining. The membrane was blocked in 2.5% skimmed milk in TBS-T (TBS + 0.05% Tween-20) for 1 hr at room temperature with agitation. Primary antibody was incubated for 1 hr at room temperature with agitation. We used [a competitor's] secondary 1:3000) for 1 hr at room temperature with agitation. We washed with TBST three times after primary and secondary antibody, each wash lasting for 5-10 mins. ECL-plus (Amersham) was used rather than ECL, which is considerably more sensitive. Final detection was on autoradiography film. I hope this information helps. Please do not hesitate to contact me should you require further assistance.