Please I want to block ChREBP Ab (NB400-135) using ChREBP peptide I need help to no the procedure and how much I need to add from the peptide to the ChREBP Ab

Name: CHREBP Antibody Blocking Peptide
Brand: Novus Biologicals
SKU: NB400-135PEP
Rating: 4 (1 reviews)

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Here is a protocol for a blocking peptide control for a western blot: Materials and Reagents Blocking buffer (usually TBST plus either 5% non-fat dry milk or 3% BSA for Western blot, or PBS plus 1% BSA for IHC) - Antibody - Blocking (immunizing) peptide - Two tubes - Two identical samples (e.g. a Western blot with two identical lanes, cut in half; two slides containing the cells of interest; etc) Method 1. Determine the optimal concentration of antibody that consistently gives a positive result in your particular protocol. Using that concentration, determine how much antibody you will need for two experiments. a. For example, an antibody is being used successfully in Western blot at 0.5 ug/ml. You will need 2 ml of antibody solution to stain one strip of a Western blot. Thus, you would use 1 ug of antibody in 2 ml buffer for each strip. 2. Dilute the necessary amount of antibody in blocking buffer to the final volume needed for the two experiments. Divide this equally into two tubes. 3. In the first tube, labeled locked, add the blocking peptide to a final concentration of 1 ug/ml (2 ug total peptide in this example). In the second tube, labeled Control, add an equivalent amount of buffer. 4. Incubate both tubes, with agitation, at room temperature for 30 minutes, or overnight at 4 degrees C. 5. Perform the staining protocol on the two identical samples, using the blocked antibody for one and the control for the other. Be careful not to mix up the strips using the blocked and control antibodies! 6. Observe the staining. The staining that disappears when using the blocked antibody is specific to the antibody. (See note i) Notes i. If more than one band disappears in Western blot by peptide/antigen competition, those bands contain the antigenic determinants and could be fragments of the full antigen or a complex containing the antigen.

At	=	A. thaliana	SyHa	=	Golden Syrian Hamster
All	=	All Species	Ha	=	Hamster
An	=	Animal	Hu	=	Human
ArHa	=	Armenian Hamster	I	=	Insect
Av	=	Avian	Ll	=	Llama
Bb	=	Baboon	Ma	=	Mammal
Ba	=	Bacteria	Mk	=	Monkey
Bv	=	Bovine	Mu	=	Mouse
Ca	=	Canine	Multi	=	Multi-species
Ce	=	C. Elegans	NA	=	Non-species specific
Ch	=	Chicken	Or	=	Orangutan
ChHa	=	Chinese Hamster	Pl	=	Plant
Cm	=	Cynomolgus Monkey	Pm	=	Primate
Do	=	Donkey	Po	=	Porcine
Dr	=	Drosophilia	Rb	=	Rabbit
Ec	=	E. Coli	Rt	=	Rat
Eq	=	Equine	RM	=	Rhesus Macaque
Fe	=	Feline	Sh	=	Sheep
Ft	=	Ferret	Vb	=	Vertebrate
Fi	=	Fish	Vi	=	Virus
Fu	=	Fungi	Xp	=	Xenopus
Ge	=	Gerbil	Ye	=	Yeast
GP	=	Guinea Pig	Ze	=	Zebrafish
Gt	=	Goat

Reactivity	HuSpecies Glossary
Applications	AC
Concentration	1.0 mg/ml

Description	A MLXIPL antibody blocking peptide.
Specificity	This peptide is specific for NB400-135 only.
Protein/Peptide Type	Antibody Blocking Peptide
Gene	MLXIPL

Storage	Store at -80C. Avoid freeze-thaw cycles.
Buffer	Peptide dissolved in dH2O. Contains no BSA.
Preservative	No Preservative
Concentration	1.0 mg/ml

	Ruth Ann Veach 05/25/2012
Application:	WB
Species:	Human

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