DYKDDDDK tag Polyclonal antibody (Binds to FLAG® tag epitope)

The antibody works fine in WB in the range fo 1:1000-1:5000 dilution in 5% milk TBST. In the image the samples are HEK293 lysates on a tricine SDS PAGE with primary antiFLAG antibody 1:5000 overnight at 4 ºC. It is true that the antibody could show considerable nonspecific background depending on the concentrations and cells used. In my experience, works perfectly fine and is quite sensitive in HEK293 cells, although in my EndoC-ßH1 cells signal is weaker and backgorund is very notable. However, I assume it is due to the EndoC cells, which are a pain for many applications. I also used this antibody for some IF experiments. In the EndoC line, no matter the antibody concentration, I always get non specific signal. I'm worried the antibody binds to something in particular in these cells. In HEK293 cells however, I managed to obtain good results for overexpression of a FLAG tagged peptide, same as the one on Western Blot

• Applications: Western Blot, Immunofluorescence
• Primary Antibody Dilution: For WB 1:2000 and for IF 1:1000
• Cell Tissue Type: HEK293

Also used in a pul down assay for FLAG tag plasmids

• Applications: Western Blot, Immunoprecipitation, Protein purification

This antibody worked very well in both ECL and Li-Cor procedures.

• Applications: Western Blot
• Primary Antibody Dilution: 1:1,000
• Cell Tissue Type: HeLa cells and HEK293 cells transfected with plasmids encoding Flag-tagged proteins

Good for western blot of knock-in mice but not good for immunostaining.

• Applications: Western Blot
• Cell Tissue Type: Mouse Heart

This antibody is extremely concentrated and works very well for detecting FLAG-tagged proteins in U2OS and HEK293 cells.

• Applications: Western Blot
• Primary Antibody Dilution: 1:4000
• Cell Tissue Type: HEK293 and U2OS cells

It is Ok to use it in WB but with some non-specific bands.

• Applications: Western Blot
• Primary Antibody Dilution: 1:2000
• Cell Tissue Type: primary cortical neuron infected with FLAG-fusion protein expressing lentivirus

Antibody used on HEK293T transfected with a FLAG tagged protein and western blots compared with 3 other FLAG antibodies. This one gives the strongest signal for the tagged protein (which is a about 34 kDa) so it might be very useful for poorly expressed proteins. However it also has the most background bands, with 2 main additional background bands (but with a lower intensity than my tagged protein of interest): one at around 50 kDa (just above 47 kDa marker) and another one between 118 and 85 kDa markers. So this antibody is worth a try depending on the size of your protein of interest. It gives the strongest signal of the 4 antibodies tested.

• Applications: Western Blot
• Primary Antibody Dilution: 1:1000
• Cell Tissue Type: Transfected HEK293T cell line

Very strong signal, but with some weak non specific bands.

• Applications: Western Blot

HEK293 cells were transfected with gene of interested. Primary antibody incubation was performed at 4C for an overnight and Alexa Fluor 488 secondary antibody incubation was performed at room temperature for an hour. Cells were stained with Hoechst for DNA/ nuclei . Images were taken from Zeiss LSM780 microscope.

• Applications: Western Blot, Immunohistochemistry
• Primary Antibody Dilution: 1:500
• Cell Tissue Type: Transfected HEK293

For western blots, this FLAG antibody is comparable to Sigma's M2 FLAG antibody, which has been the gold standard for FLAG.

• Applications: Western Blot
• Primary Antibody Dilution: 1:1000
• Cell Tissue Type: HEK293T

Great antibody for Western blotting. I used it at 1:1000 and got a super strong signal. Will use at a lower concentration next time.

• Applications: Western Blot,
• Primary Antibody Dilution: 1:1000
• Cell Tissue Type: HEK293T cells

Works really well for both IP of overexpressed FLAG-tagged proteins and WB!

• Applications: Western Blot, Immunoprecipitation,
• Primary Antibody Dilution: 1:2000
• Cell Tissue Type: 293T overexpressing FLAG-tagged construct

Flag tagged p53 expressed in H1299 cells.

• Applications: Western Blot,
• Primary Antibody Dilution: 1:1000
• Cell Tissue Type: H1299

Works better than another brand I had tested.

• Applications: Western Blot,
• Primary Antibody Dilution: 1:1000

This antibody has worked well for western blotting as well as immunoprecipitation. The lots have been consistent so far, we will definitely be ordering more.

• Applications: Western Blot, Immunoprecipitation,
• Primary Antibody Dilution: 1:1000
• Cell Tissue Type: H1299

Works very good for WB at 1:1000 and IP at 1:200.

• Applications: Western Blot, Immunoprecipitation,
• Primary Antibody Dilution: 1:1000
• Cell Tissue Type: Human cell lines

This is working very well for me. I ran out of another brand and got this one because I saw you had it and it was a better value. After testing it in the lab, it works just as well, if not better than the previous one. A very strong signal in my experiments.

• Applications: Western Blot,
• Primary Antibody Dilution: 1/2000

SDS-PAGE:10 ug/ul RIPA lysate of whole cells4-12% Bis-tris gradient gelTransfer:Immobilon-FL transfer membranes (Millipore) O/N at 30V, 4CBlocking:SEA Block Blocking Buffer 1hPrimary Ab:DDDDK pRb Cat# 20543-1-APO/N incubation at 4CSecondary Ab:IRDye 800CW Goat anti-Rabbit1 h incubation at room temperatureLines on WB:1. BioRad Precision Plus Protein standards2. Whole cell lysate of HEK293t cells (negative transfection)3. Whole cell lysate of HEK293t cells transfected with FLAG-tag construct 27 kDa3. Whole cell lysate of HEK293t cells transfected with FLAG-tag construct 40 kDa

• Applications: Western Blot,
• Primary Antibody Dilution: 1:1000
• Cell Tissue Type: HEK293t

• Applications: Western Blot, Immunoprecipitation,
• Cell Tissue Type: 10T1/2 cells