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LOADING CONTROL ANTIBODIES FOR WESTERN BLOTTING by Deborah Grainger The proteins and peptides that regularly serve as endogenous or internal controls may not always take center stage in your research, but they are indispensable to your conducting meaningful experiments and are essential for publication. Western blotting requires such controls: it is widely used for the semi-quantification of protein levels under of a set of different experimental parameters. Protein standards are required to make sense of Western blotting results, and check that any increases and decreases in target proteins are actually due to experimental manipulations and not, for example, because the sample went wandering during gel loading. Internal control proteins — i.e. those with constant, unchanging levels — are usually detected in a second round of blotting, following primary detection of your protein of interest. This step is used to standardize results and normalize for any errors that creep into a Western blot experiment, such as sample loss through loading at SDS-PAGE or Western blot transfer. The loading control candidates for Western blotting are usually proteins with high and constitutive expression. As mentioned above, the most basic criterion for a loading control is that its level remains unchanged throughout an experiment, regardless of tissues or cell types used and how they are handled. This means control candidates require careful selection; even bastions of the loading control repertoire — such as β-actin and α-tubulin — can be affected by the conditions of your experiment (so be sure to double check your chosen manipulations do not impact them.) Here we’ve provided background information on each of the internal control proteins targeted by control antibodies we offer, helping you choose the best control for your personal needs… Actin Type: Whole cell/cytoplasm Molecular weight: ~42kDa The six isofo ms of actin constitute a family of highly conse ved globula p oteins comp ised of th ee main isofo m g oups, alpha, beta, and gamma. The alpha actins alpha C1 and alpha 1 and 2 a e a majo constituent of the cont actile appa atus in muscle tissues. The beta (β) and gamma 1 and 2 (γ1 and γ2) actins co exist in most cell types and a e an integ al pa t of the cytoskeleton; they a e mediato s of cell t afficking, st uctu al integ ity and cell motility. Togethe , actins a e the most abundant p oteins in the typical euka yotic cell, accounting fo about 15 pe cent of total p otein in some cell types. As such, actin is widely used as an inte nal cont ol in Weste n blotting expe iments. P oteintech’s polyclonal ACTB antibody (20536 1 AP) was gene ated using a β actin p otein antigen (amino acids 14 167) and ecognizes all fo ms of actin, making it a pan actin antibody. Many studies use β actin antibodies ecognizing all actin isofo ms to p obe fo this loading cont ol collective. Howeve , if you studies involve wo k with skeletal muscle samples, o you a e wo king with conditions that see changes in cell g owth o alte ed inte actions with the ext acellula mat ix, anothe loading cont ol may be bette suited to you needs. Actin antibodies Related antibodies ACTB 10081 224 Rabbit poly ACTA1 10082 472 Rabbit poly ACTB 10081 974 Mouse Mono ACTA2 10155 850 Rabbit poly HeLa cell lysate (10 ug/lane) was sepa ated by SDS PAGE and actin was detected by anti ACTB antibody 20536 1 AP at va ying dilutions. (L R) 1:500, 1:1,000, 1:2,000 and 1:4,000. LOADING CONTROL ANTIBODIES FOR WESTERN BLOTTING COX-4 Type: Mitochondrial Molecular weight: 17kDa COX 4, o COX V (cytoch ome c oxidase subunit V), is a nuclea encoded subunit of the human mitochond ial espi ato y chain enzyme cytoch ome c oxidase (COX). The COX 4 subunit can be exp essed as eithe of two isofo ms, isofo m 1 and 2 named COX4 1 and COX4 2 espectively. COX4 1 exp ession is ubiquitous th oughout all tissues, whilst COX4 2 is lung specific. Because of its dependably high level, COX4 1 is commonly detected as an effective loading cont ol fo mitochond ial p oteins. Howeve , some caution is advised when selecting this p otein fo Weste n blot detection as many othe p oteins un at its 17kDa size du ing SDS PAGE (make su e you band of inte est won’t be obscu ed). t is also adviso y to double check that any expe imental manipulations do not affect its levels. Fo an alte native mitochond ial p otein loading cont ol see ou ent y on VDAC1 below. P oteintech’s COX4 1 antibody (11242 1 AP) was gene ated against a COX4 1 whole p otein antigen (amino acids 1 169) and also ecognizes COX4 2. mmunop ecipitation of COXV f om mouse skeletal muscle whole tissue lysate using COXV antibody 11242 1 AP. COX-4 antibodies COX411 10085 086 Rabbit poly COX412 10085 058 Rabbit poly GAPDH Type: Whole Cell/cytoplasmic Molecular weight: 36kDa Glyce aldehyde 3 phosphate dehyd ogenase, commonly known as GAPDH, catalyzes the sixth step of glycolysis. t also pa ticipates in nuclea events such as t ansc iption, RNA binding and t anspo t, DNA eplication and epai , as well as apoptosis. ts exp ession is high and constant in most tissues and cell types, ea ning the gene and its p otein housekeeping status. Because of this, GAPDH is commonly used as a p otein loading cont ol in Weste n blot and inte nal cont ol fo RT PCR. Howeve , the amount of GAPDH exp ession can diffe between some tissues and its suitability fo you expe iment should be taken into conside ation befo e selecting this ta get as a cont ol. t is also wo th noting that some physiological facto s, such as hypoxia and diabetes, inc ease GAPDH exp ession in ce tain cell and tissue types. P oteintech has both monoclonal GAPDH (60004 1 g) and polyclonal GAPDH (10494 1 AP) antibodies available, both aised against a whole p otein antigen (amino acids 1 335) of human o igin. GAPDH antibodies GAPDH 10087 386 Rabbit poly GAPDH 10087 384 Mouse poly Weste n blot with Hela cell lysate using anti GAPDH (10494 1 AP) at va ious dilutions (L R: 1:2000, 1:4000, 1:8000 and 1:16000). Lamin B1 Type: Nuclear Molecular weight: 66kDa Lamins a e integ al components of the nuclea lamina, a dense, fib ous laye unde lying the nuclea envelope on its nucleoplasmic side. Lamins play an impo tant ole in the st uctu al integ ity of the nucleus and its t affic cont ol, as well as inte acting with ch omatin and gene exp ession. Ve teb ate lamins consist of two types, A and B. The LMNB1 gene encodes one of the two B type p oteins, lamin B1 and can be used as a loading cont ol fo those wo king with nuclea f actions; howeve , this p otein is not suitable fo samples whe e the nuclea envelope has been emoved. t is also wo thwhile to note that lamins become phospho ylated du ing mitosis when the lamina mat ix is eve sibly disassembled. P oteintech’s LMNB1 antibody (12987 1 AP) has been aised against a p otein antigen (amino acids 236 to 586 at the C te minus) and is validated fo use in Weste n blot, HC, EL SA and immunofluo escence. mmunofluo escence analysis of Lamin B1 in HepG2 cells, using LMNB1 antibody 12987 1 AP at 1:50 dilution and F TC labeled donkey anti abbit gG ( ed). Lamin antibodies Lmnb1 Related antibodies 10089 490 Rabbit poly LMNA/C 10089 486 Rabbit poly LOADING CONTROL ANTIBODIES FOR WESTERN BLOTTING PCNA Type: Nuclear Molecular weight: 36kDa P olife ating Cell Nuclea Antigen (PCNA) is a p ocessivity facto fo DNA polyme ase δ; it helps cont ol euka yotic DNA eplication by inc easing polyme ase nucleotide p ocessing ability du ing elongation of the leading st and. PCNA p otein has been highly conse ved th oughout evolution the amino acid sequences of ats and humans diffe by only 4 of 261 amino acids meaning whole p otein aised antibodies ta geting PCNA should wo k ac oss multiple species. Levels of PCNA do not va y with cell cycle status in mammalian cells, but, as it is mo e abundant in p olife ating cells, PCNA is mostly used as loading cont ol in cell populations unde going p olife ation. PCNA is best avoided if you expe iments induce DNA damage as this p otein is quickly deg aded when DNA damage pathways a e activated. mmunofluo escence analysis of PCNA in HepG2 cells, using PCNA antibody 10205 2 AP at 1:50 dilution and F TC labeled donkey anti abbit gG (g een). P oteintech’s PCNA antibody is a abbit polyclonal antibody aised against an inte nal egion of human PCNA, encompassing amino acids 8 256. PCNA antibodies PCNA 10091 936 Rabbit poly PCNA 10091 934 Mouse poly Tubulin Type: Whole cell/cytoplasmic Molecular weight: 50-55kDa Tubulins a e the majo components of mic otubules, the majo t anspo t netwo k in cells. The mic otubules a e involved in a wide va iety of cellula activities anging f om mitosis and t anspo t events to cell movement and the maintenance of cell shape. Highly and stably exp essed and conse ved ac oss the species, tubulins make excellent whole cell o cytoplasmic f action loading cont ols in Weste n blotting. Howeve , tubulin exp ession may va y acco ding to esistance to antimic obial and antimitotic d ugs. P oteintech has polyclonal antibodies against seve al tubulin subunits including an α tubulin antibody (11224 1 AP) and two β tubulin antibodies 10068 1 AP and 10094 1 Ap. Related antibodies Tubulin antibodies GAPDH 10081 874 Rabbit poly 10096 498 β tubulin(antigen: amino acids 43 258) Rabbit poly 10096 496 β tubulin (antigen: amino acids 57 294) Rabbit poly α tubulin 10082 812 Mouse Mono Weste n blot with Hela cell lysate using anti GAPDH (10494 1 AP) at va ious dilutions (L R: 1:2000, 1:4000, 1:8000 and 1:16000). TBP Type: Nuclear Molecular weight: 38kDa The TATA binding p otein (TBP) is a t ansc iption facto that binds specifically to the TATA box DNA sequence, found a ound 25 30 base pai s upst eam of the t ansc iption sta t site of a ound 10 20 pe cent of euka yotic gene p omote s. TBP, along with a va iety of TBP associated facto s, make up the TF D, a gene al t ansc iption facto that in tu n makes up pa t of the RNA polyme ase p einitiation complex (P C). As one of the few p oteins in the RNA P C that binds DNA in a sequence specific manne , it helps position RNA polyme ase ove the t ansc iption sta t site of the gene. TBP is widely exp essed, though its levels a e highest in the testis and ova y. This p otein is not a suitable standa d fo expe iments whe e DNA and othe nuclea components have been emoved. P oteintech has a polyclonal antibody available, aised against full length TBP binding p otein. TBP antibodies COLO 320 cells we e subjected to SDS PAGE followed by weste n blot with 22006 1 AP(TBP antibody) at dilution of 1:1000 TBP 10081 148 Rabbit poly LOADING CONTROL ANTIBODIES FOR WESTERN BLOTTING VDCA1/Porin Type: Mitochondrial Molecular weight : 31kDa Voltage-dependent anion-selective channel protein 1 (VDAC1), also named porin 31HM, porin 31HL or plasmalemmal porin, is the most widely expressed isoform of the eukaryotic mitochondrial porin family. It forms a channel through the outer mitochondrial membrane which adopts an open conformation at low or zero membrane potential and a closed conformation at potentials above 30-40mV. VDAC1 is generally thought to be the principal means by which metabolites diffuse in and out of the mitochondria and also has a secondary role in apoptotic signaling. It is ubiquitously expressed throughout tissues, though its levels are elevated in heart, liver and skeletal muscle tissues. It is conserved across the species including, chimpanzee, dog, cow, mouse, rat, chicken, and zebrafish. Detection of VDAC1 is a suitable mitochondrial loading control in whole-cell, cytoplasmic or mitochondrial extracts. Proteintech has two rabbit polyclonal antibodies against VDAC1 available. HEK293 cell lysate was sepa ated by SDS PAGE followed by detection of VDAC1 by Weste n blotting with P oteintech VDAC1 antibody 10866 1 AP at a dilution of 1:1000. VDAC1 antibodies VDAC1 10096 950 Rabbit poly VDAC1 10096 948 Mouse poly (Both aised against a whole p otein antigen: VDAC1 amino acid esidues 1 283.) 0615 Lit. No. 161294W