Anti halotag pab

Manufactured by Promega

Sourced in Japan, Germany

The Anti-HaloTag pAb is a polyclonal antibody that specifically recognizes the HaloTag protein. The HaloTag protein is a genetically engineered protein tag that can be fused to a target protein for the purpose of visualization, purification, or immobilization. The Anti-HaloTag pAb can be used to detect the presence of the HaloTag protein in various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry.

Automatically generated - may contain errors

Lab products found in correlation

Close spelling variants of this product

Anti-HaloTag (22 citations)

4 protocols using anti halotag pab

Detecting Click Chemistry Reactions

Check if the same lab product or an alternative is used in the 5 most similar protocols

For SPAAC, cell pellets were lysed by sonication in 100 µL 1% SDS/SPAAC quench buffer. For iEDDA, cell pellets were lysed by sonication in 100 µL 1% SDS/iEDDA quench buffer. Protein concentrations were determined with a BCA protein assay (Thermo-Fisher) and cell lysates were normalized by protein concentration. Samples were prepared in 1× LDS sample buffer (Life Technologies), with 10 µg protein loaded per well, and separated by SDS-PAGE on NuPage 4–12% Bis-Tris gels in MES running buffer (Life Technologies). TAMRA-fluorescence was analyzed on a Typhoon variable mode imager (GE Healthsciences) using a TAMRA filter. Gels were then transferred to nitrocellulose using iBLOT (Life Technologies), blocked in Odyssey blocking buffer (LiCor) for 1 h at RT, and incubated in anti-HaloTag pAb (Promega, G9281) at 1:2000 overnight in TBST. Membranes were washed 3 times in TBST, and incubated in anti-rabbit IRDye 800CW (LiCor) at 1:10,000 in TBST for 1 h at RT. Membranes were washed three times in TBST and imaged on the Odyssey Infrared Imager (LiCor).

Murrey H.E., Judkins J.C., am Ende C.W., Ballard T.E., Fang Y., Riccardi K., Di L., Guilmette E.R., Schwartz J.W., Fox J.M, & Johnson D.S. (2015). Systematic Evaluation of Bioorthogonal Reactions in Live Cells with Clickable HaloTag Ligands: Implications for Intracellular Imaging. Journal of the American Chemical Society, 137(35), 11461-11475.

+ Open protocol

+ Expand

Quantitative Proteomic Analysis of SPAAC and iEDDA

Check if the same lab product or an alternative is used in the 5 most similar protocols

For SPAAC, cell pellets were lysed by sonication
in 100 μL 1% SDS/SPAAC quench buffer. For iEDDA, cell pellets
were lysed by sonication in 100 μL 1% SDS/iEDDA quench buffer.
Protein concentrations were determined with a BCA protein assay (Thermo-Fisher)
and cell lysates were normalized by protein concentration. Samples
were prepared in 1× LDS sample buffer (Life Technologies), with
10 μg protein loaded per well, and separated by SDS-PAGE on
NuPage 4–12% Bis-Tris gels in MES running buffer (Life Technologies).
TAMRA-fluorescence was analyzed on a Typhoon variable mode imager
(GE Healthsciences) using a TAMRA filter. Gels were then transferred
to nitrocellulose using iBLOT (Life Technologies), blocked in Odyssey
blocking buffer (LiCor) for 1 h at RT, and incubated in anti-HaloTag
pAb (Promega, G9281) at 1:2000 overnight in TBST. Membranes were washed
3 times in TBST, and incubated in goat anti-rabbit IRDye 800CW (LiCor)
at 1:10,000 in TBST for 1 h at RT. Membranes were washed three times
in TBST and imaged on the Odyssey Infrared Imager (LiCor).

+ Open protocol

+ Expand

Immunocytochemistry Visualization of Intracellular Proteins

Check if the same lab product or an alternative is used in the 5 most similar protocols

For immunocytochemistry, cells on coverslips were fixed in 4% paraformaldehyde-phosphate buffer for 10 min at 48 h after transfection, and then washed three times briefly in PBS. To study intracellular protein localization, cells were permeabilized for 20 min at room temperature with 0.1% Triton X-100 (SIGMA, Kanagawa, Japan) in PBS, blocked with 3% Goat serum (Gibco, Thermo Fisher, MA, USA) in PBS for 20 min, and incubated for 1 h at room temperature with a 1:200 dilution of AntiHalo-Tag pAb (Promega, Tokyo, Japan). Cells were washed three times with PBS, and then incubated for 1 h at room temperature with a 1:200 dilution of the secondary antibody, Alexa Fluor 546 goat anti-rabbit antibody. Total actin was stained with Alexa Fluor 488 phalloidin (Invitrogen, Thermo Fisher, MA, USA), and a 1/1000 dilution of DAPI (KPL, MA, USA) to visualize the nucleus. Cells were washed in PBS 3 times, and coverslips were mounted onto a slide glass with Pro-long Gold Antifade Mountant (Invitrogen, Thermo Fisher, MA, USA). Images were taken with an Olympus Fluoview FV-10i system (Olympus, Okaya, Japan).

Miyajima H., Moteki H., Day T., Nishio S.Y., Murata T., Ikezono T., Takeda H., Abe S., Iwasaki S., Takahashi M., Naito Y., Yamazaki H., Kanda Y., Kitajiri S.I, & Usami S.I. (2020). Novel ACTG1 mutations in patients identified by massively parallel DNA sequencing cause progressive hearing loss. Scientific Reports, 10, 7056.

+ Open protocol

+ Expand

Titin Reactivity Analysis by SDS-PAGE

Cited 2 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

To evaluate titin reactivity for quality control and experimental success, fiber samples treated with or without TEV protease were analyzed by agarose-strengthened 1.8–2.4% SDS–PAGE and visualized with Coomassie blue, as previously done (Prado et al., 2005 (link); Unger et al., 2017 (link)). Relative band intensities between intact and cut N2A titin were used to measure % of total titins cut. Western blotting was performed as described elsewhere (Unger et al., 2017 (link)). To detect HaloTag-TEV titin, we used the primary antibody (anti-HaloTag pAB, Promega 6928) and secondary antibody (anti-rabbit HRP, Acris, Herford, Germany, R1364HRP). Signals from HRP‐conjugated secondary antibodies were visualized by chemiluminescence (Amersham ECL start Western blotting detection reagent, GE Healthcare) and recorded using the ImageQuant LAS 4000 Imaging System (GE Healthcare). Signal intensity was quantified by densitometry using the ImageQuant TL software (GE Healthcare). A subset of TEV-treated fibers (n = 5/genotype) were also loaded onto Coomassie-stained, agarose-strengthened, 2.4% SDS–PAGE, titin gels to measure the MyHC:titin, titin:NEB, and MyHC:NEB protein ratios after active contraction.

Li Y., Hessel A.L., Unger A., Ing D., Recker J., Koser F., Freundt J.K, & Linke W.A. (2020). Graded titin cleavage progressively reduces tension and uncovers the source of A-band stability in contracting muscle. eLife, 9, e64107.

+ Open protocol

+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!