Anti-HIV1 tat antibody (ab14029)

Could you give me the conditions under which the Western blot on the datasheet was run?

ANSWER:

Thank you for your enquiry.

Below please find the protocol used for blotting with this antibody. The antibody dilution was 1:2000, and recombinant fusion protein used as the antigen.

I hope this information helps. Please do not hesitate to contact us if you need anything further.

Protocol for Western Blot Performed with IgY

Materials 1. Tris-buffered Saline-Tween 20 solution (TBS-T): 1.21 g Tris-base (10 mM), 8.77 g NaCl (150 mM), in 1 L of H20, pH 7.4, containing 0.05% (v/v) Tween-20. 2. Non-fat dry milk. 3. HRP-conjugated anti-IgY secondary. 4. Immun-blot colorimetric assay kit (Bio-Rad Catalog #: 170-8235).

Method 1. Separate appropriate amount of cell lysates (1-10 ul of 0.5 mg/ml each lane) by 10-20% SDS-PAGE, followed by transferring to PVDF membrane. 2. Block membrane with 5% non-fat milk in TBS-T (Tris-buffered saline containing 0.05% Tween, pH 7.4) for 1 hour at room temperature or longer at 4C. (BSA is not recommended as a blocking reagent). 3. Rinse membrane with TBS-T. 4. Incubate membrane with IgY antibodies appropriately diluted with 5% milk in TBS-T @ R.T. for 1 h. 5. Wash membrane with TBS-T, 3 min each, total of 3 times. 6. Incubate with 2nd antibody @ R.T. for 1 h. 7. Wash with TBS-T, 3-5 min each with shaking, total of 3 times. 8. Perform color development. Or perform ECL detection of signal using Pierce ECL kit.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM High background - cells that were not treated with the tat protein are staining positive for tat

SAMPLE cultured human fetal neurons

PRIMARY ANTIBODY chicken anti-HIV-tat at 1:200 (recommended) in blocking solution overnight, washed 3x5min with PBS-

SECONDARY ANTIBODY Abcam goat anti-chicken Cy5 at 1:500 for 1hour, washed 3x5min with PBS-

DETECTION METHOD Leica confocal

POSITIVE AND NEGATIVE CONTROLS USED Had several conditions with no tat treatment with same staining as tat-treated. Secondary antibody only was clean - no background.

ANTIBODY STORAGE CONDITIONS Aliquoted and stored at -20

FIXATION OF SAMPLE cold 70% ethanol over night

ANTIGEN RETRIEVAL n/a

PERMEABILIZATION STEP n/a

BLOCKING CONDITIONS 1% Ig-free BSA, 0.5% gelatin (from fish skin), 2% filtered horse serum, 2%human serum, 2.5mM EDTA, in PBS-, blocked overnight at 4degrees

HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Haven't yet tried it, but was thinking about pre-absorbing the antibody with either fixed coverslips of neurons (not tat treated) or with neuron lysate to get rid of non-specific binding. Was looking for advice in that direction...

ANSWER:

I'm sorry to hear you are having a problem with ab14029, thank you for taking the time to fill in the questionnaire, it is very useful.

I would like to suggest the following modifications to your protocol: -fix your cells for 5-10min in ice cold acetone or methanol (a longer fixation may be responsible for the non specific binding) -block one hour in 5% BSA (you may wish to try your blocking solution but we tend to recommend 5%BSA in PBST or PBS) -incubate your primary antibody overnight in PBS (or PBST) overnight at 1:200, try a lower dilution (maybe 1:100).

Please let me know if this helps and do not hesitate to contact us if you still have problems,

I am interested in the HIV1 tat antibody (ab14029), however, I was wondering from what HIV viral strain was the tat protein generated? If you don't have the particular strain, do you know from what country the virus originated?

ANSWER:

We don't know the viral strain and the source of virus. However, this antibody should recognize tat from various stains since this is a polyclonal antibody against the full length protein.