產品名稱：RAB-IP Anti-rabbit IgG (HRP) for IP/CoIP

貨號： IF9203

品牌：Engibody

簡介：IP/CoIP專用二抗，消除抗體分子輕重鏈干擾

RAB-IP™ IP專用的抗兔IgG二抗（天然構象完整分子抗體特異性）（HRP偶聯）是一種蛋白IP-WB試劑，其能夠無障礙地檢測來自上游IP/CoIP的蛋白質印跡目標蛋白帶，而不受變性IgG的干擾（IP一抗）。這允許在不受IgG重鏈（50kDa）和輕鏈（25kDa）干擾的情況下檢測（共）免疫沉淀蛋白。通常，這種干擾傾向于源于傳統的二抗，其識別在免疫沉淀過程中與抗原一起釋放的一抗或來自裂解物本身的內源性IgG的輕鏈和重鏈。

RAB-IP™ 如果免疫沉淀的抗原-抗體復合物在下游WB之前被還原和變性，則用于IP的抗兔IgG（HRP標記）僅識別天然（非還原）一抗（來自兔），因此重鏈和輕鏈的條帶不會顯示。

描述

RAB-IP™ IP專用的抗兔IgG二抗（天然構象完整分子抗體特異性）（HRP偶聯）

反應性

識別兔IgG完整抗體分子

經過測試的應用實驗

WB:1/1000-1/2000。最佳稀釋度應由最終用戶確定。

特異性

該二抗對天然兔rabbit多克隆/單克隆抗體完整分子具有特異性，同時不識別兔IgG抗體單獨輕重鏈

免疫原

以天然兔IgG為免疫原，在山羊身上制備抗體，然后制備只識別兔抗體完整分子同時不識別兔抗體單獨輕重鏈的單克隆抗體。

克隆性

單克隆，克隆號：3B8

同型對照

山羊IgG

類型

抗體在10 mM PBS中，pH 7.4，50%甘油，10 mg/mL（1%w/v）BSA作為穩定劑，0.01%（w/v）硫柳汞作為防腐劑。

存儲說明

儲存于-20°C，避免冷凍/解凍循環。不要分裝抗體。

偶聯標記

辣根過氧化物酶（HRP）

抗體來源宿主

山羊

RAB-IP™ Anti- rabbit IgG for IP (HRP Conjugated) only recognize native (non-reduced) primary antibodies (from rabbit) and therefore the bands of heavy and light chains is highly minimized, if the immunoprecipitated antigen-antibody complex is fully reduced and denatured before downstream WB.

• Description

• RAB-IP™ Anti- rabbit IgG (native conformation antibody specific) for IP (HRP Conjugated)

• Reactivity

• Rabbit

• Tested applications

• WB : 1/1000-1/2000. Optimal dilutions should be determined by the end user.

• Specificity

• This secondary antibody is specific to native rabbit polyclonal/monoclonal antibody

• Immunogen

• The antibody was developed in goat using the nature rabbit IgG as the immunogen and then develop monoclonal antibody.

• Clonality

• Monoclonal, clone number: 3B8

• Isotype

• Goat IgG

• Form

• The antibody in 10 mM phosphate buffered saline (PBS), pH 7.4, 50% glycerol with 10 mg/mL (1% w/v) BSA (IgG free, protease free) as a stabilizer and 0.01% (w/v) thimerosal as preservative.

• Storage instruction

• Store at -20°C, Avoid freeze / thaw cycle. Do not aliquot the antibody.

• Conjugation

• Horseradish Peroxidase (HRP)

• Host

• Goat

Figure 1. RAB-IP™ Anti- rabbit IgG for IP (HRP) secondary antibody images: Western blotting.

IP conditions:

target protein TBP was immunoprecipitated from 0.5 mL cell lysate of 1x107 Hela cells with 5 µg anti-human TBP rabbit monoclonal antibody and protein A/G agarose beads (Engibody, IF0001).

WB conditions:

the immunoprecipitated antigen-antibody complex should be boiled for 5-10 minutes in SDS sample buffer with a increase in SDS amount if required, make sure that the complex is fully reduced and denatured before downstream WB.

then electrophoresis, transferred to a PVDF membrane, and incubate with an anti- TBP rabbit monoclonal antibody.

Secondary Antibody Detection:

Lane 1: Detection with RAB-IP™ Anti-rabbit IgG for IP (HRP) (CAT: IF9203)

The heavy and light chains can not be seen, confirming that although the immunoprecipitating heavy and light-chains are present, detects only native antibody and not denatured heavy and light-chains.

Lane 2: Detection with a traditional goat anti-rabbit IgG (H&L) (HRP) secondary antibody (CAT: AT0097)

Figure 2. A schematic representation of IP-WB workflow by using RAB-IP™ secondary antibody.

Using primary antibody coupled beads

RAB-IP™ Anti-rabbit IgG for IP (HRP Conjugated) only recognize native (non-reduced) primary antibodies (from rabbit) and therefore the bands of heavy chain and light chain are highly minimized, if the immunoprecipitated antigen-antibody complex is fully reduced and denatured before downstream WB.

Figure 3. A schematic representation of IP-WB workflow by using RAB-IP™ secondary antibody.

Using protein A/G Beads + primary antibody

RAB-IP™ Anti-rabbit IgG for IP (HRP Conjugated) only recognize native (non-reduced) primary antibodies (from rabbit) and therefore the bands of heavy chain and light chain are highly minimized, if the immunoprecipitated antigen-antibody complex is fully reduced and denatured before downstream WB.